Angiopoietin-like 3 (ANGPTL3) iRNA compositions and methods of use thereof

ABSTRACT

The invention relates to double-stranded ribonucleic acid (dsRNA) compositions targeting the ANGPTL3 gene, as well as methods of inhibiting expression of ANGPTL3 and methods of treating subjects having a disorder of lipid metabolism, such as hyperlipidemia or hypertriglyceridemia, using such dsRNA compositions.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.17/089,854, filed on Nov. 5, 2020, which is a continuation of U.S.patent application Ser. No. 16/411,261, filed on May 14, 2019, now U.S.Pat. No. 10,934,545, issued on Mar. 2, 2021, which is a continuation ofU.S. patent application Ser. No. 15/683,999, filed on Aug. 23, 2017, nowU.S. Pat. No. 10,337,010, issued on Jul. 2, 2019, which is acontinuation of U.S. patent application Ser. No. 15/068,912 filed onMar. 14, 2016, now U.S. Pat. No. 9,771,591, issued on Sep. 26, 2017,which is a continuation of U.S. patent application Ser. No. 14/132,999filed on Dec. 18, 2013, now U.S. Pat. No. 9,322,018, issued on Apr. 26,2016, which is a 35 U.S.C. 111(a) continuation application, which claimspriority to PCT/US2012/043378, filed on Jun. 20, 2012, U.S. ProvisionalApplication No. 61/499,620, filed on Jun. 21, 2011, and to U.S.Provisional Application No. 61/638,288, filed on Apr. 25, 2012. Theentire contents of each of the foregoing applications are herebyincorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Apr. 5, 2022, isnamed 121301_00316_SL.txt and is 444,511 bytes in size.

BACKGROUND OF THE INVENTION

Angiopoietin-like 3 (ANGPTL3) is a member of the angiopoietin-likefamily of secreted factors that regulates lipid metabolism and that ispredominantly expressed in the liver (Koishi, R. et al., (2002) Nat.Genet. 30(2):151-157). ANGPTL3 dually inhibits the catalytic activitiesof lipoprotein lipase (LPL), which catalyzes the hydrolysis oftriglycerides, and of endothelial lipase (EL), which hydrolyzes highdensity lipoprotein (HDL) phospholipids. In hypolipidemic, yet obese,KK/Snk mice, a reduction in ANGPTL3 expression has a protective effectagainst hyperlipidemia and artherosclerosis by promoting the clearanceof triglycerides (Ando et al., (2003) J. Lipid Res., 44:1216-1223).Human ANGPTL3 plasma concentrations positively correlate with plasma HDLcholesterol and HDL phospholipid levels (Shimamura et al., (2007)Arterioscler. Thromb. Vasc. Biol., 27:366-372).

Disorders of lipid metabolism can lead to elevated levels of serumlipids, such as triglycerides and/or cholesterol. Elevated serum lipidsare strongly associated with high blood pressure, cardiovasculardisease, diabetes and other pathologic conditions. Hypertriglyceridemiais an example of a lipid metabolism disorder that is characterized byhigh blood levels of triglycerides. It has been associated withatherosclerosis, even in the absence of high cholesterol levels(hypercholesterolemia). When triglyceride concentrations are excessive(i.e., greater than 1000 mg/dl or 12 mmol/1), hypertriglyceridemia canalso lead to pancreatitis. Hyperlipidemia is another example of a lipidmetabolism disorder that is characterized by elevated levels of any oneor all lipids and/or lipoproteins in the blood. Current treatments fordisorders of lipid metabolism, including dieting, exercise and treatmentwith statins and other drugs, are not always effective. Accordingly,there is a need in the art for alternative treatments for subjectshaving disorders of lipid metabolism.

SUMMARY OF THE INVENTION

The present invention provides iRNA compositions which effect theRNA-induced silencing complex (RISC)-mediated cleavage of RNAtranscripts of an ANGPL3 gene. The ANGPL3 gene may be within a cell,e.g., a cell within a subject, such as a human. The present inventionalso provides methods of using the iRNA compositions of the inventionfor inhibiting the expression of an ANGPL3 gene and/or for treating asubject who would benefit from inhibiting or reducing the expression ofan ANGPL3 gene, e.g., a subject suffering or prone to suffering from adisorder of lipid metabolism, such as a subject suffering or prone tosuffering from hyperlipidemia or hypertriglyceridemia.

Accordingly, in one aspect, the present invention providesdouble-stranded ribonucleic acids (dsRNAs) for inhibiting expression ofANGPTL3. The dsRNAs comprise a sense strand and an antisense strand,wherein the sense strand comprises at least 15 contiguous nucleotidesdiffering by no more than 3 nucleotides from the nucleotide sequence ofSEQ ID NO:1 and the antisense strand comprises at least 15 contiguousnucleotides differing by no more than 3 nucleotides from the nucleotidesequence of SEQ ID NO:5.

In another aspect, the present invention provides double-strandedribonucleic acids (dsRNAs) for inhibiting expression of ANGPTL3. ThedsRNAs comprise a sense strand and an antisense strand, the antisensestrand comprising a region of complementarity which comprises at least15 contiguous nucleotides differing by no more than 3 nucleotides fromany one of the antisense sequences listed in Tables 2, 3, 7, 8, 9 and10.

In one embodiment, the sense and antisense strands comprise sequencesselected from the group consisting of AD-53063.1, AD-53001.1,AD-53015.1, AD-52986.1, AD-52981.1, AD-52953.1, AD-53024.1, AD-53033.1,AD-53030.1, AD-53080.1, AD-53073.1, AD-53132.1, AD-52983.1, AD-52954.1,AD-52961.1, AD-52994.1, AD-52970.1, AD-53075.1, AD-53147.1, AD-53077.1of Tables 7 and 8.

In certain embodiments of the invention, the dsRNAs comprise at leastone modified nucleotide. In one embodiment, at least one of the modifiednucleotides is selected from the group consisting of a 2′-O-methylmodified nucleotide, a nucleotide comprising a 5′-phosphorothioategroup, and a terminal nucleotide linked to a cholesteryl derivative or adodecanoic acid bisdecylamide group. In another embodiment, the modifiednucleotide is selected from the group consisting of a 2′-deoxy-2′-fluoromodified nucleotide, a 2′-deoxy-modified nucleotide, a lockednucleotide, an abasic nucleotide, a 2′-amino-modified nucleotide, a2′-alkyl-modified nucleotide, a morpholino nucleotide, aphosphoramidate, and a non-natural base comprising nucleotide.

The region of complementarity of the dsRNAs may be at least 17nucleotides in length, between 19 and 21 nucleotides in length, or 19nucleotides in length.

In one embodiment, each strand of a dsRNA is no more than 30 nucleotidesin length.

At least one strand of a dsRNA may comprise a 3′ overhang of at least 1nucleotide or at least 2 nucleotides.

In certain embodiments, a dsRNA further comprises a ligand. In oneembodiment, the ligand is conjugated to the 3′ end of the sense strandof the dsRNA.

In some embodiments, the ligand is one or more N-acetylgalactosamine(GalNAc) derivatives attached through a bivalent or trivalent branchedlinker. In particular embodiments, the ligand is

In some embodiments, the RNAi agent is conjugated to the ligand as shownin the following schematic

In some embodiments, the RNAi agent further includes at least onephosphorothioate or methylphosphonate internucleotide linkage. In someembodiments, the phosphorothioate or methylphosphonate internucleotidelinkage is at the 3′-terminal of one strand. In some embodiments, thestrand is the antisense strand. In other embodiments, the strand is thesense strand.

In one embodiment, the region of complementarity of a dsRNA consists ofone of the antisense sequences of Tables 2, 3, 7, 8, 9 and 10.

In another embodiment, a dsRNA comprises a sense strand consisting of asense strand sequence selected from the sequences of Tables 2, 3, 7, 8,9 and 10, and an antisense strand consisting of an antisense sequenceselected from the sequences of Tables 2, 3, 7, 8, 9 and 10.

In another aspect, the present invention provides a cell, e.g., ahepatocyte, containing a dsRNA of the invention.

In yet another aspect, the present invention provides a vector encodingat least one strand of a dsRNA, wherein the dsRNA comprises a region ofcomplementarity to at least a part of an mRNA encoding ANGPTL3, whereinthe dsRNA is 30 base pairs or less in length, and wherein the dsRNAtargets the mRNA for cleavage. The region of complementarity may beleast 15 nucleotides in length or 19 to 21 nucleotides in length.

In a further aspect, the present invention provides a cell comprising avector encoding at least one strand of a dsRNA, wherein the dsRNAcomprises a region of complementarity to at least a part of an mRNAencoding ANGPTL3, wherein the dsRNA is 30 base pairs or less in length,and wherein the dsRNA targets the mRNA for cleavage.

In one aspect, the present invention provides a pharmaceuticalcomposition for inhibiting expression of an ANGPTL3 gene comprising adsRNA or vector of the invention.

In one embodiment, the pharmaceutical composition comprises a lipidformulation, such as a MC3, SNALP or XTC formulation.

In another aspect, the present invention provides methods of inhibitingANGPTL3 expression in a cell. The methods include contacting the cellwith a dsRNA or a vector of the invention, and maintaining the cellproduced for a time sufficient to obtain degradation of the mRNAtranscript of an ANGPTL3 gene, thereby inhibiting expression of theANGPTL3 gene in the cell.

The cell may be within a subject, such as a human subject, for example ahuman subject suffering from a disorder of lipid metabolism, e.g.,hyperlipidemia or hypertriglyceridemia.

In one embodiment of the methods of the invention, ANGPTL3 expression isinhibited by at least about 30%, at least about 35%, at least about 40%,at least about 45%, at least about 50%, at least about 55%, at leastabout 60%, at least about 65%, at least about 70%, at least about 75%,at least about 80%, at least about 85%, at least about 90%, at leastabout 91%, at least about 92%, at least about 93%, at least about 94%,at least about 95%, at least about 96%, at least about 97%, at leastabout 98%, or at least about 99%.

In another aspect, the present invention provides methods of treating asubject having a disorder that would benefit from reduction in ANGPTL3expression, e.g., a disorder of lipid metabolism, such as hyperlipidemiaor hypertriglyceridemia. The methods include administering to thesubject a therapeutically effective amount of a dsRNA or a vector of theinvention, thereby treating the subject.

The disorder may be disorder of lipid metabolism, such as hyperlipidemiaor hypertriglyceridemia

In one embodiment, the administration of the dsRNA to the subject causesa decrease in the level of a serum lipid, triglycerides, cholesteroland/or free fatty acids; and/or a decrease in ANGPTL3 proteinaccumulation. In one embodiment, administration of the dsRNA to thesubject causes a decrease in the level of LDL-C, HDL-C, VLDL-C, IDL-Cand/or total cholesterol.

In one embodiment, the dsRNA is administered at a dose of about 0.01mg/kg to about 10 mg/kg, e.g., about 0.05 mg/kg to about 5 mg/kg, about0.05 mg/kg to about 10 mg/kg, about 0.1 mg/kg to about 5 mg/kg, about0.1 mg/kg to about 10 mg/kg, about 0.2 mg/kg to about 5 mg/kg, about 0.2mg/kg to about 10 mg/kg, about 0.3 mg/kg to about 5 mg/kg, about 0.3mg/kg to about 10 mg/kg, about 0.4 mg/kg to about 5 mg/kg, about 0.4mg/kg to about 10 mg/kg, about 0.5 mg/kg to about 5 mg/kg, about 0.5mg/kg to about 10 mg/kg, about 1 mg/kg to about 5 mg/kg, about 1 mg/kgto about 10 mg/kg, about 1.5 mg/kg to about 5 mg/kg, about 1.5 mg/kg toabout 10 mg/kg, about 2 mg/kg to about about 2.5 mg/kg, about 2 mg/kg toabout 10 mg/kg, about 3 mg/kg to about 5 mg/kg, about 3 mg/kg to about10 mg/kg, about 3.5 mg/kg to about 5 mg/kg, about 4 mg/kg to about 5mg/kg, about 4.5 mg/kg to about 5 mg/kg, about 4 mg/kg to about 10mg/kg, about 4.5 mg/kg to about 10 mg/kg, about 5 mg/kg to about 10mg/kg, about 5.5 mg/kg to about 10 mg/kg, about 6 mg/kg to about 10mg/kg, about 6.5 mg/kg to about 10 mg/kg, about 7 mg/kg to about 10mg/kg, about 7.5 mg/kg to about 10 mg/kg, about 8 mg/kg to about 10mg/kg, about 8.5 mg/kg to about 10 mg/kg, about 9 mg/kg to about 10mg/kg, or about 9.5 mg/kg to about 10 mg/kg. Values and rangesintermediate to the recited values are also intended to be part of thisinvention.

For example, the dsRNA may be administered at a dose of about 0.01,0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5,0.6, 0.7. 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8. 1.9, 2,2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8. 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5,3.6, 3.7, 3.8. 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8. 4.9, 5,5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8. 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5,6.6, 6.7, 6.8. 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8. 7.9, 8,8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8. 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5,9.6, 9.7, 9.8. 9.9, or about 10 mg/kg. Values and ranges intermediate tothe recited values are also intended to be part of this invention.

In another embodiment, the dsRNA is administered at a dose of about 0.5to about 50 mg/kg, about 0.75 to about 50 mg/kg, about 1 to about 50mg/mg, about 1.5 to about 50 mg/kb, about 2 to about 50 mg/kg, about 2.5to about 50 mg/kg, about 3 to about 50 mg/kg, about 3.5 to about 50mg/kg, about 4 to about 50 mg/kg, about 4.5 to about 50 mg/kg, about 5to about 50 mg/kg, about 7.5 to about 50 mg/kg, about 10 to about 50mg/kg, about 15 to about 50 mg/kg, about 20 to about 50 mg/kg, about 20to about 50 mg/kg, about 25 to about 50 mg/kg, about 25 to about 50mg/kg, about 30 to about 50 mg/kg, about 35 to about 50 mg/kg, about 40to about 50 mg/kg, about 45 to about 50 mg/kg, about 0.5 to about 45mg/kg, about 0.75 to about 45 mg/kg, about 1 to about 45 mg/mg, about1.5 to about 45 mg/kb, about 2 to about 45 mg/kg, about 2.5 to about 45mg/kg, about 3 to about 45 mg/kg, about 3.5 to about 45 mg/kg, about 4to about 45 mg/kg, about 4.5 to about 45 mg/kg, about 5 to about 45mg/kg, about 7.5 to about 45 mg/kg, about 10 to about 45 mg/kg, about 15to about 45 mg/kg, about 20 to about 45 mg/kg, about 20 to about 45mg/kg, about 25 to about 45 mg/kg, about 25 to about 45 mg/kg, about 30to about 45 mg/kg, about 35 to about 45 mg/kg, about 40 to about 45mg/kg, about 0.5 to about 40 mg/kg, about 0.75 to about 40 mg/kg, about1 to about 40 mg/mg, about 1.5 to about 40 mg/kb, about 2 to about 40mg/kg, about 2.5 to about 40 mg/kg, about 3 to about 40 mg/kg, about 3.5to about 40 mg/kg, about 4 to about 40 mg/kg, about 4.5 to about 40mg/kg, about 5 to about 40 mg/kg, about 7.5 to about 40 mg/kg, about 10to about 40 mg/kg, about 15 to about 40 mg/kg, about 20 to about 40mg/kg, about 20 to about 40 mg/kg, about 25 to about 40 mg/kg, about 25to about 40 mg/kg, about 30 to about 40 mg/kg, about 35 to about 40mg/kg, about 0.5 to about 30 mg/kg, about 0.75 to about 30 mg/kg, about1 to about 30 mg/mg, about 1.5 to about 30 mg/kb, about 2 to about 30mg/kg, about 2.5 to about 30 mg/kg, about 3 to about 30 mg/kg, about 3.5to about 30 mg/kg, about 4 to about 30 mg/kg, about 4.5 to about 30mg/kg, about 5 to about 30 mg/kg, about 7.5 to about 30 mg/kg, about 10to about 30 mg/kg, about 15 to about 30 mg/kg, about 20 to about 30mg/kg, about 20 to about 30 mg/kg, about 25 to about 30 mg/kg, about 0.5to about 20 mg/kg, about 0.75 to about 20 mg/kg, about 1 to about 20mg/mg, about 1.5 to about 20 mg/kb, about 2 to about 20 mg/kg, about 2.5to about 20 mg/kg, about 3 to about 20 mg/kg, about 3.5 to about 20mg/kg, about 4 to about 20 mg/kg, about 4.5 to about 20 mg/kg, about 5to about 20 mg/kg, about 7.5 to about 20 mg/kg, about 10 to about 20mg/kg, or about 15 to about 20 mg/kg. Values and ranges intermediate tothe recited values are also intended to be part of this invention.

For example, subjects can be administered a therapeutic amount of iRNA,such as about 0.5, 0.6, 0.7. 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6,1.7, 1.8. 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8. 2.9, 3, 3.1,3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8. 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6,4.7, 4.8. 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8. 5.9, 6, 6.1,6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8. 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6,7.7, 7.8. 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8. 8.9, 9, 9.1,9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8. 9.9, 10.5, 11, 11.5, 12, 12.5, 13,13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or about 50 mg/kg.Values and ranges intermediate to the recited values are also intendedto be part of this invention.

In another aspect, the present invention provides methods of inhibitingthe expression of ANGPTL3 in a subject. The methods includeadministering to the subject a therapeutically effective amount of adsRNA or a vector of the invention, thereby inhibiting the expression ofANGPTL3 in the subject.

In yet another aspect, the invention provides kits for performing themethods of the invention. In one aspect, the invention provides a kitfor performing a method of inhibiting expression of ANGPTL3 gene in acell by contacting a cell with a double stranded RNAi agent in an amounteffective to inhibit expression of the ANGPTL3 in the cell. The kitcomprises an RNAi agent and instructions for use and, optionally, meansfor administering the RNAi agent to a subject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic of the experimental procedure used for in vivotests described in Example 2.

FIG. 2A is a graph showing measured levels of ANGPTL3 protein in WT miceafter treatment with the indicated iRNA or a control.

FIG. 2B is a graph showing measured levels of ANGPTL3 proten in ob/obmice after treatment with the indicated iRNA or a control.

FIG. 3A is a graph showing measured levels of LDL-c in WT mice aftertreatment with the indicated iRNA or a control.

FIG. 3B is a graph showing measured levels of LDL-c in ob/ob mice aftertreatment with the indicated iRNA or a control.

FIG. 4A is a graph showing measured levels of triglycerides in WT miceafter treatment with the indicated iRNA or a control.

FIG. 4B is a graph showing measured levels of triglycerides in ob/obmice after treatment with the indicated iRNA or a control.

FIG. 5A is a graph showing measured levels of total cholesterol (TC) inWT mice after treatment with the indicated iRNA or a control.

FIG. 5B is a graph showing measured levels of total cholesterol (TC) inob/ob mice after treatment with the indicated iRNA or a control.

FIG. 6A is a graph showing measured levels of HDL-c in WT mice aftertreatment with the indicated iRNA or a control.

FIG. 6B is a graph showing measured levels of HDL-c in ob/ob mice aftertreatment with the indicated iRNA or a control.

FIG. 7 is a graph showing measured levels of ANGPTL3 protein in humanPCS transgenic mice after treatment with a single dose of the indicatediRNA or a control.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides iRNA compositions, which effect theRNA-induced silencing complex (RISC)-mediated cleavage of RNAtranscripts of an ANGPTL3gene. The ANGPTL3 gene may be within a cell,e.g., a cell within a subject, such as a human. The present inventionalso provides methods of using the iRNA compositions of the inventionfor inhibiting the expression of an ANGPTL3gene and/or for treating asubject having a disorder that would benefit from inhibiting or reducingthe expression of an ANGPTL3gene, e.g., a disorder of lipid metabolism,such as hyperlipidemia or hypertriglyceridemia.

The iRNAs of the invention include an RNA strand (the antisense strand)having a region which is about 30 nucleotides or less in length, e.g.,15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21,15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25,18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26,19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27,20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27,21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length, which regionis substantially complementary to at least part of an mRNA transcript ofan ANGPTL3 gene. The use of these iRNAs enables the targeted degradationof mRNAs of an ANGPTL3 gene in mammals. Very low dosages of ANGPTL3iRNAs, in particular, can specifically and efficiently mediate RNAinterference (RNAi), resulting in significant inhibition of expressionof an ANGPTL3 gene. Using cell-based assays, the present inventors havedemonstrated that iRNAs targeting ANGPTL3 can mediate RNAi, resulting insignificant inhibition of expression of an ANGPTL3 gene. Thus, methodsand compositions including these iRNAs are useful for treating a subjectwho would benefit by a reduction in the levels and/or activity of anANGPTL3 protein, such as a subject having a disorder of lipidmetabolism, such as hyperlipidemia or hypertriglyceridemia.

The following detailed description discloses how to make and usecompositions containing iRNAs to inhibit the expression of an ANGPTL3gene, as well as compositions and methods for treating subjects havingdiseases and disorders that would benefit from inhibition and/orreduction of the expression of this gene.

I. Definitions

In order that the present invention may be more readily understood,certain terms are first defined. In addition, it should be noted thatwhenever a value or range of values of a parameter are recited, it isintended that values and ranges intermediate to the recited values arealso intended to be part of this invention.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element, e.g., a plurality of elements.

The term “including” is used herein to mean, and is used interchangeablywith, the phrase “including but not limited to”. The term “or” is usedherein to mean, and is used interchangeably with, the term “and/or,”unless context clearly indicates otherwise.

The term “ANGPTL3” refers to an angiopoietin like protein 3 having anamino acid sequence from any vertebrate or mammalian source, including,but not limited to, human, bovine, chicken, rodent, mouse, rat, porcine,ovine, primate, monkey, and guinea pig, unless specified otherwise. Theterm also refers to fragments and variants of native ANGPTL3 thatmaintain at least one in vivo or in vitro activity of a native ANGPTL3.The term encompasses full-length unprocessed precursor forms of ANGPTL3as well as mature forms resulting from post-translational cleavage ofthe signal peptide and forms resulting from proteolytic processing ofthe fibrinogen-like domain. The sequence of a human ANGPTL3 mRNAtranscript can be found at, for example, GenBank Accession No. GI:41327750 (NM_014495.2; SEQ ID NO:1). The predicted sequence of rhesusANGPTL3 mRNA can be found at, for example, GenBank Accession No. GI:297278846 (XM_001086114.2; SEQ ID NO:2). The sequence of mouse ANGPTL3mRNA can be found at, for example, GenBank Accession No. GI: 142388354(NM_013913.3; SEQ ID NO:3). The sequence of rat ANGPTL3 mRNA can befound at, for example, GenBank Accession No. GI: 68163568(NM_001025065.1; SEQ ID NO:4).

The term “ANGPTL3” as used herein also refers to a particularpolypeptide expressed in a cell by naturally occurring DNA sequencevariations of the ANGPTL3 gene, such as a single nucleotide polymorphismin the ANGPTL3 gene. Numerous SNPs within the ANGPTL3 gene have beenidentified and may be found at, for example, NCBI dbSNP (see, e.g.,www.ncbi.nlm.nih.gov/snp). Non-limiting examples of SNPs within theANGPTL3 gene may be found at, NCBI dbSNP Accession Nos. rs193064039;rs192778191; rs192764027; rs192528948; rs191931953; rs191293319;rs191171206; rs191145608; rs191086880; rs191012841; or rs190255403.

As used herein, “target sequence” refers to a contiguous portion of thenucleotide sequence of an mRNA molecule formed during the transcriptionof an ANGPTL3gene, including mRNA that is a product of RNA processing ofa primary transcription product. In one embodiment, the target portionof the sequence will be at least long enough to serve as a substrate foriRNA-directed cleavage at or near that portion of the nucleotidesequence of an mRNA molecule formed during the transcription of anANGPTL3gene.

The target sequence may be from about 9-36 nucleotides in length, e.g.,about 15-30 nucleotides in length. For example, the target sequence canbe from about 15-30 nucleotides, 15-29, 15-28, 15-27, 15-26, 15-25,15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29,18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30,19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20,20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21,21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22nucleotides in length. Ranges and lengths intermediate to the aboverecited ranges and lengths are also contemplated to be part of theinvention.

As used herein, the term “strand comprising a sequence” refers to anoligonucleotide comprising a chain of nucleotides that is described bythe sequence referred to using the standard nucleotide nomenclature.

“G,” “C,” “A,” “T” and “U” each generally stand for a nucleotide thatcontains guanine, cytosine, adenine, thymidine and uracil as a base,respectively. However, it will be understood that the term“ribonucleotide” or “nucleotide” can also refer to a modifiednucleotide, as further detailed below, or a surrogate replacementmoiety. The skilled person is well aware that guanine, cytosine,adenine, and uracil can be replaced by other moieties withoutsubstantially altering the base pairing properties of an oligonucleotidecomprising a nucleotide bearing such replacement moiety. For example,without limitation, a nucleotide comprising inosine as its base can basepair with nucleotides containing adenine, cytosine, or uracil. Hence,nucleotides containing uracil, guanine, or adenine can be replaced inthe nucleotide sequences of dsRNA featured in the invention by anucleotide containing, for example, inosine. In another example, adenineand cytosine anywhere in the oligonucleotide can be replaced withguanine and uracil, respectively to form G-U Wobble base pairing withthe target mRNA. Sequences containing such replacement moieties aresuitable for the compositions and methods featured in the invention.

The terms “iRNA”, “RNAi agent,” “iRNA agent,”, “RNA interference agent”as used interchangeably herein, refer to an agent that contains RNA asthat term is defined herein, and which mediates the targeted cleavage ofan RNA transcript via an RNA-induced silencing complex (RISC) pathway.iRNA directs the sequence-specific degradation of mRNA through a processknown as RNA interference (RNAi). The iRNA modulates, e.g., inhibits,the expression of ANGPTL3 in a cell, e.g., a cell within a subject, suchas a mammalian subject.

In one embodiment, an RNAi agent of the invention includes a singlestranded RNA that interacts with a target RNA sequence, e.g., an ANGPTL3target mRNA sequence, to direct the cleavage of the target RNA. Withoutwishing to be bound by theory, long double stranded RNA introduced intocells is broken down into siRNA by a Type III endonuclease known asDicer (Sharp et al., Genes Dev. 2001, 15:485). Dicer, aribonuclease-III-like enzyme, processes the dsRNA into 19-23 base pairshort interfering RNAs with characteristic two base 3′ overhangs(Bernstein, et al., (2001) Nature 409:363). The siRNAs are thenincorporated into an RNA-induced silencing complex (RISC) where one ormore helicases unwind the siRNA duplex, enabling the complementaryantisense strand to guide target recognition (Nykanen, et al., (2001)Cell 107:309). Upon binding to the appropriate target mRNA, one or moreendonucleases within the RISC cleave the target to induce silencing(Elbashir, et al., (2001) Genes Dev. 15:188). Thus, in one aspect theinvention relates to a single stranded RNA (siRNA) generated within acell and which promotes the formation of a RISC complex to effectsilencing of the target gene, i.e., an ANGPTL3 gene. Accordingly, theterm “siRNA” is also used herein to refer to an RNAi as described above.

In another aspect, the RNAi agent is a single-stranded antisense RNAmolecule. An antisense RNA molecule is complementary to a sequencewithin the target mRNA. Antisense RNA can inhibit translation in astoichiometric manner by base pairing to the mRNA and physicallyobstructing the translation machinery, see Dias, N. et al., (2002) Mol.Cancer Ther. 1:347-355. The single-stranded antisense RNA molecule maybe about 13 to about 30 nucleotides in length and have a sequence thatis complmentary to a target sequence. For example, the single-strandedantisense RNA molecule may comprise a sequence that is at least about13, 14, 15, 16, 17, 18, 19, 20, or more contiguous nucleotides from oneof the antisense sequences in Tables 2, 3, 7, 8, 9 and 10.

In another embodiment, an “iRNA” for use in the compositions and methodsof the invention is a double-stranded RNA and is referred to herein as a“double stranded RNAi agent,” “double-stranded RNA (dsRNA) molecule,”“dsRNA agent,” or “dsRNA”. The term “dsRNA”, refers to a complex ofribonucleic acid molecules, having a duplex structure comprising twoanti-parallel and substantially complementary nucleic acid strands,referred to as having “sense” and “antisense” orientations with respectto a target RNA, i.e., an ANGPTL3 gene. In some embodiments of theinvention, a double-stranded RNA (dsRNA) triggers the degradation of atarget RNA, e.g., an mRNA, through a post-transcriptional gene-silencingmechanism referred to herein as RNA interference or RNAi.

The duplex region may be of any length that permits specific degradationof a desired target RNA through a RISC pathway, and may range from about9 to 36 base pairs in length, e.g., about 15-30 base pairs in length,for example, about 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 base pairsin length, such as about 15-30, 15-29, 15-28, 15-27, 15-26, 15-25,15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29,18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30,19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20,20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21,21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 basepairs in length. Ranges and lengths intermediate to the above recitedranges and lengths are also contemplated to be part of the invention.

The two strands forming the duplex structure may be different portionsof one larger RNA molecule, or they may be separate RNA molecules. Wherethe two strands are part of one larger molecule, and therefore areconnected by an uninterrupted chain of nucleotides between the 3′-end ofone strand and the 5′-end of the respective other strand forming theduplex structure, the connecting RNA chain is referred to as a “hairpinloop.” A hairpin loop can comprise at least one unpaired nucleotide. Insome embodiments, the hairpin loop can comprise at least 2, at least 3,at least 4, at least 5, at least 6, at least 7, at least 8, at least 9,at least 10, at least 20, at least 23 or more unpaired nucleotides.

Where the two substantially complementary strands of a dsRNA arecomprised by separate RNA molecules, those molecules need not, but canbe covalently connected. Where the two strands are connected covalentlyby means other than an uninterrupted chain of nucleotides between the3′-end of one strand and the 5′-end of the respective other strandforming the duplex structure, the connecting structure is referred to asa “linker.” The RNA strands may have the same or a different number ofnucleotides. The maximum number of base pairs is the number ofnucleotides in the shortest strand of the dsRNA minus any overhangs thatare present in the duplex. In addition to the duplex structure, an RNAimay comprise one or more nucleotide overhangs.

As used herein, the term “nucleotide overhang” refers to at least oneunpaired nucleotide that protrudes from the duplex structure of an iRNA,e.g., a dsRNA. For example, when a 3′-end of one strand of a dsRNAextends beyond the 5′-end of the other strand, or vice versa, there is anucleotide overhang. A dsRNA can comprise an overhang of at least onenucleotide; alternatively the overhang can comprise at least twonucleotides, at least three nucleotides, at least four nucleotides, atleast five nucleotides or more. A nucleotide overhang can comprise orconsist of a nucleotide/nucleoside analog, including adeoxynucleotide/nucleoside. The overhang(s) can be on the sense strand,the antisense strand or any combination thereof. Furthermore, thenucleotide(s) of an overhang can be present on the 5′-end, 3′-end orboth ends of either an antisense or sense strand of a dsRNA.

In one embodiment, the antisense strand of a dsRNA has a 1-10nucleotide, e.g., a 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide,overhang at the 3′-end and/or the 5′-end. In one embodiment, the sensestrand of a dsRNA has a 1-10 nucleotide, e.g., a 1, 2, 3, 4, 5, 6, 7, 8,9, or 10 nucleotide, overhang at the 3′-end and/or the 5′-end. Inanother embodiment, one or more of the nucleotides in the overhang isreplaced with a nucleoside thiophosphate.

The terms “blunt” or “blunt ended” as used herein in reference to adsRNA mean that there are no unpaired nucleotides or nucleotide analogsat a given terminal end of a dsRNA, i.e., no nucleotide overhang. One orboth ends of a dsRNA can be blunt. Where both ends of a dsRNA are blunt,the dsRNA is said to be blunt ended. To be clear, a “blunt ended” dsRNAis a dsRNA that is blunt at both ends, i.e., no nucleotide overhang ateither end of the molecule. Most often such a molecule will bedouble-stranded over its entire length.

The term “antisense strand” or “guide strand” refers to the strand of aniRNA, e.g., a dsRNA, which includes a region that is substantiallycomplementary to a target sequence, e.g., an ANGPTL3 mRNA. As usedherein, the term “region of complementarity” refers to the region on theantisense strand that is substantially complementary to a sequence, forexample a target sequence, e.g., an ANGPTL3 nucleotide sequence, asdefined herein. Where the region of complementarity is not fullycomplementary to the target sequence, the mismatches can be in theinternal or terminal regions of the molecule. Generally, the mosttolerated mismatches are in the terminal regions, e.g., within 5, 4, 3,or 2 nucleotides of the 5′- and/or 3′-terminus of the iRNA.

The term “sense strand” or “passenger strand” as used herein, refers tothe strand of an iRNA that includes a region that is substantiallycomplementary to a region of the antisense strand as that term isdefined herein.

As used herein, and unless otherwise indicated, the term“complementary,” when used to describe a first nucleotide sequence inrelation to a second nucleotide sequence, refers to the ability of anoligonucleotide or polynucleotide comprising the first nucleotidesequence to hybridize and form a duplex structure under certainconditions with an oligonucleotide or polynucleotide comprising thesecond nucleotide sequence, as will be understood by the skilled person.Such conditions can, for example, be stringent conditions, wherestringent conditions can include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mMEDTA, 50° C. or 70° C. for 12-16 hours followed by washing (see, e.g.,“Molecular Cloning: A Laboratory Manual, Sambrook, et al. (1989) ColdSpring Harbor Laboratory Press). Other conditions, such asphysiologically relevant conditions as can be encountered inside anorganism, can apply. The skilled person will be able to determine theset of conditions most appropriate for a test of complementarity of twosequences in accordance with the ultimate application of the hybridizednucleotides.

Complementary sequences within an iRNA, e.g., within a dsRNA asdescribed herein, include base-pairing of the oligonucleotide orpolynucleotide comprising a first nucleotide sequence to anoligonucleotide or polynucleotide comprising a second nucleotidesequence over the entire length of one or both nucleotide sequences.Such sequences can be referred to as “fully complementary” with respectto each other herein. However, where a first sequence is referred to as“substantially complementary” with respect to a second sequence herein,the two sequences can be fully complementary, or they can form one ormore, but generally not more than 5, 4, 3 or 2 mismatched base pairsupon hybridization for a duplex up to 30 base pairs, while retaining theability to hybridize under the conditions most relevant to theirultimate application, e.g., inhibition of gene expression via a RISCpathway. However, where two oligonucleotides are designed to form, uponhybridization, one or more single stranded overhangs, such overhangsshall not be regarded as mismatches with regard to the determination ofcomplementarity. For example, a dsRNA comprising one oligonucleotide 21nucleotides in length and another oligonucleotide 23 nucleotides inlength, wherein the longer oligonucleotide comprises a sequence of 21nucleotides that is fully complementary to the shorter oligonucleotide,can yet be referred to as “fully complementary” for the purposesdescribed herein.

“Complementary” sequences, as used herein, can also include, or beformed entirely from, non-Watson-Crick base pairs and/or base pairsformed from non-natural and modified nucleotides, in so far as the aboverequirements with respect to their ability to hybridize are fulfilled.Such non-Watson-Crick base pairs include, but are not limited to, G:UWobble or Hoogstein base pairing.

The terms “complementary,” “fully complementary” and “substantiallycomplementary” herein can be used with respect to the base matchingbetween the sense strand and the antisense strand of a dsRNA, or betweenthe antisense strand of an iRNA agent and a target sequence, as will beunderstood from the context of their use.

As used herein, a polynucleotide that is “substantially complementary toat least part of” a messenger RNA (mRNA) refers to a polynucleotide thatis substantially complementary to a contiguous portion of the mRNA ofinterest (e.g., an mRNA encoding ANGPTL3). For example, a polynucleotideis complementary to at least a part of an ANGPTL3mRNA if the sequence issubstantially complementary to a non-interrupted portion of an mRNAencoding ANGPTL3.

In general, the majority of nucleotides of each strand areribonucleotides, but as described in detail herein, each or both strandscan also include one or more non-ribonucleotides, e.g., adeoxyribonucleotide and/or a modified nucleotide. In addition, an “iRNA”may include ribonucleotides with chemical modifications. Suchmodifications may include all types of modifications disclosed herein orknown in the art. Any such modifications, as used in an iRNA molecule,are encompassed by “iRNA” for the purposes of this specification andclaims.

The term “inhibiting,” as used herein, is used interchangeably with“reducing,” “silencing,” “downregulating,” “suppressing” and othersimilar terms, and includes any level of inhibition.

The phrase “inhibiting expression of an ANGPTL3,” as used herein,includes inhibition of expression of any ANGPTL3 gene (such as, e.g., amouse ANGPTL3 gene, a rat ANGPTL3 gene, a monkey ANGPTL3 gene, or ahuman ANGPTL3 gene) as well as variants or mutants of an ANGPTL3 genethat encode an ANGPTL3 protein.

“Inhibiting expression of an ANGPTL3 gene” includes any level ofinhibition of an ANGPTL3 gene, e.g., at least partial suppression of theexpression of an ANGPTL3 gene, such as an inhibition by at least about5%, at least about 10%, at least about 15%, at least about 20%, at leastabout 25%, at least about 30%, at least about 35%, at least about 40%,at least about 45%, at least about 50%, at least about 55%, at leastabout 60%, at least about 65%, at least about 70%, at least about 75%,at least about 80%, at least about 85%, at least about 90%, at leastabout 91%, at least about 92%, at least about 93%, at least about 94%,at least about 95%, at least about 96%, at least about 97%, at leastabout 98%, or at least about 99%.

The expression of an ANGPTL3 gene may be assessed based on the level ofany variable associated with ANGPTL3 gene expression, e.g., ANGPTL3 mRNAlevel or ANGPTL3 protein level. The expression of an ANGPTL3 may also beassessed indirectly based on the levels of a serum lipid, atriglyceride, cholesterol (including LDL-C, HDL-C, VLDL-C, IDL-C andtotal cholesterol), or free fatty acids. Inhibition may be assessed by adecrease in an absolute or relative level of one or more of thesevariables compared with a control level. The control level may be anytype of control level that is utilized in the art, e.g., a pre-dosebaseline level, or a level determined from a similar subject, cell, orsample that is untreated or treated with a control (such as, e.g.,buffer only control or inactive agent control).

In one embodiment, at least partial suppression of the expression of anANGPTL3 gene, is assessed by a reduction of the amount of ANGPTL3 mRNAwhich can be isolated from or detected in a first cell or group of cellsin which an ANGPTL3 gene is transcribed and which has or have beentreated such that the expression of an ANGPTL3 gene is inhibited, ascompared to a second cell or group of cells substantially identical tothe first cell or group of cells but which has or have not been sotreated (control cells). The degree of inhibition may be expressed interms of:

$\frac{( {{mRNA}{in}{control}{cells}} ) - ( {{mRNA}{in}{treated}{cells}} )}{( {{mRNA}{in}{control}{cells}} )}{\bullet 100}\%$

The phrase “contacting a cell with an RNAi agent,” such as a dsRNA, asused herein, includes contacting a cell by any possible means.Contacting a cell with an RNAi agent includes contacting a cell in vitrowith the iRNA or contacting a cell in vivo with the iRNA. The contactingmay be done directly or indirectly. Thus, for example, the RNAi agentmay be put into physical contact with the cell by the individualperforming the method, or alternatively, the RNAi agent may be put intoa situation that will permit or cause it to subsequently come intocontact with the cell.

Contacting a cell in vitro may be done, for example, by incubating thecell with the RNAi agent. Contacting a cell in vivo may be done, forexample, by injecting the RNAi agent into or near the tissue where thecell is located, or by injecting the RNAi agent into another area, e.g.,the bloodstream or the subcutaneous space, such that the agent willsubsequently reach the tissue where the cell to be contacted is located.For example, the RNAi agent may contain and/or be coupled to a ligand,e.g., GalNAc3, that directs the RNAi agent to a site of interest, e.g.,the liver. Combinations of in vitro and in vivo methods of contactingare also possible. For example, a cell may also be contacted in vitrowith an RNAi agent and subsequently transplanted into a subject.

In one embodiment, contacting a cell with an iRNA includes “introducing”or “delivering the iRNA into the cell” by facilitating or effectinguptake or absorption into the cell. Absorption or uptake of an iRNA canoccur through unaided diffusive or active cellular processes, or byauxiliary agents or devices. Introducing an iRNA into a cell may be invitro and/or in vivo. For example, for in vivo introduction, iRNA can beinjected into a tissue site or administered systemically. In vivodelivery can also be done by a beta-glucan delivery system, such asthose described in U.S. Pat. Nos. 5,032,401 and 5,607,677, and U.S.Publication No. 2005/0281781, the entire contents of which are herebyincorporated herein by reference. In vitro introduction into a cellincludes methods known in the art such as electroporation andlipofection. Further approaches are described herein below and/or areknown in the art.

The term “SNALP” refers to a stable nucleic acid-lipid particle. A SNALPis a vesicle of lipids coating a reduced aqueous interior comprising anucleic acid such as an iRNA or a plasmid from which an iRNA istranscribed. SNALPs are described, e.g., in U.S. Patent ApplicationPublication Nos. 20060240093, 20070135372, and in InternationalApplication No. WO 2009082817, the entire contents of which are herebyincorporated herein by reference. Examples of “SNALP” formulations aredescribed below.

As used herein, a “subject” is an animal, such as a mammal, including aprimate (such as a human, a non-human primate, e.g., a monkey, and achimpanzee), a non-primate (such as a cow, a pig, a camel, a llama, ahorse, a goat, a rabbit, a sheep, a hamster, a guinea pig, a cat, a dog,a rat, a mouse, a horse, and a whale), or a bird (e.g., a duck or agoose). In an embodiment, the subject is a human, such as a human beingtreated or assessed for a disease, disorder or condition that wouldbenefit from reduction in ANGPTL3 expression; a human at risk for adisease, disorder or condition that would benefit from reduction inANGPTL3 expression; a human having a disease, disorder or condition thatwould benefit from reduction in ANGPTL3 expression; and/or human beingtreated for a disease, disorder or condition that would benefit fromreduction in ANGPTL3 expression as described herein. As used herein, theterms “treating” or “treatment” refer to a beneficial or desired resultincluding, such as lowering levels of triglycerides in a subject. Theterms “treating” or “treatment” also include, but are not limited to,alleviation or amelioration of one or more symptoms of a disorder oflipid metabolism, such as, e.g., a decrease in the size of eruptivexanthomas. “Treatment” can also mean prolonging survival as compared toexpected survival in the absence of treatment.

By “lower” in the context of a disease marker or symptom is meant astatistically significant decrease in such level. The decrease can be,for example, at least 10%, at least 20%, at least 30%, at least 40% ormore, and is preferably down to a level accepted as within the range ofnormal for an individual without such disorder. As used herein,“prevention” or “preventing,” when used in reference to a disease,disorder or condition thereof, that would benefit from a reduction inexpression of an ANGPTL3 gene, refers to a reduction in the likelihoodthat a subject will develop a symptom associated with such disease,disorder, or condition, e.g., high triglyceride levels or eruptivexanthoma. The likelihood of developing a high tryglyceride levels oreruptive xanthoma is reduced, for example, when an individual having oneor more risk factors for a high tryglyceride levels or eruptive xanthomaeither fails to develop high tryglyceride levels or eruptive xanthoma ordevelops high tryglyceride levels or eruptive xanthoma with lessseverity relative to a population having the same risk factors and notreceiving treatment as described herein. The failure to develop adisease, disorder or condition, or the reduction in the development of asymptom associated with such a disease, disorder or condition i (e.g.,by at least about 10% on a clinically accepted scale for that disease ordisorder), or the exhibition of delayed symptoms delayed (e.g., by days,weeks, months or years) is considered effective prevention.

As used herein, the term “serum lipid” refers to any major lipid presentin the blood. Serum lipids may be present in the blood either in freeform or as a part of a protein complex, e.g., a lipoprotein complex.Non-limiting examples of serum lipids may include triglycerides andcholesterol, such as total cholesterol (TG), low density lipoproteincholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), verylow density lipoprotein cholesterol (VLDL-C) and intermediate-densitylipoprotein cholesterol (IDL-C).

As used herein, a “disorder of lipid metabolism” refers to any disorderassociated with or caused by a disturbance in lipid metabolism. Forexample, this term includes any disorder, disease or condition that canlead to hyperlipidemia, or condition characterized by abnormal elevationof levels of any or all lipids and/or lipoproteins in the blood. Thisterm refers to an inherited disorder, such as familialhypertriglyceridemia, or an acquired disorder, such as a disorderacquired as a result of a diet or intake of certain drugs. Exemplarydisorders of lipid metabolism include, but are not limited to,atherosclerosis, dyslipidemia, hypertriglyceridemia (includingdrug-induced hypertriglyceridemia, diuretic-inducedhypertriglyceridemia, alcohol-induced hypertriglyceridemia, β-adrenergicblocking agent-induced hypertriglyceridemia, estrogen-inducedhypertriglyceridemia, glucocorticoid-induced hypertriglyceridemia,retinoid-induced hypertriglyceridemia, cimetidine-inducedhypertriglyceridemia, and familial hypertriglyceridemia), acutepancreatitis associated with hypertriglyceridemia, chylomicron syndrom,familial chylomicronemia, Apo-E deficiency or resistance, LPL deficiencyor hypoactivity, hyperlipidemia (including familial combinedhyperlipidemia), hypercholesterolemia, gout associated withhypercholesterolemia, xanthomatosis (subcutaneous cholesterol deposits).

Cardiovascular diseases associated with disorders of lipid metabolismare also considered “disorders of lipid metabolism”, as defined herein.These diseases may include coronary artery disease (also called ischemicheart disease), inflammation associated with coronary artery disease,restenosis, peripheral vascular diseases, and stroke.

Disorders related to body weight are also considered “disorders of lipidmetabolism”, as defined herein. Such disorders may include obesity,metabolic syndrome including independent components of metabolicsyndrome (e.g., central obesity, FBG/pre-diabetes/diabetes,hypercholesterolemia, hypertriglyceridemia, and hypertension),hypothyroidism, uremia, and other conditions associated with weight gain(including rapid weight gain), weight loss, maintenance of weight loss,or risk of weight regain following weight loss.

Blood sugar disorders are further considered “disorders of lipidmetabolism”, as defined herein. Such disorders may include diabetes,hypertension, and polycystic ovarian syndrome related to insulinresistance. Other exemplary disorders of lipid metabolism may alsoinclude renal transplantation, nephrotic syndrome, Cushing's syndrome,acromegaly, systemic lupus erythematosus, dysglobulinemia,lipodystrophy, glycogenosis type I, and Addison's disease.

“Therapeutically effective amount,” as used herein, is intended toinclude the amount of an RNAi agent that, when administered to a subjecthaving a disorder of lipid metabolism, is sufficient to effect treatmentof the disease (e.g., by diminishing, ameliorating or maintaining theexisting disease or one or more symptoms of disease). The“therapeutically effective amount” may vary depending on the RNAi agent,how the agent is administered, the disease and its severity and thehistory, age, weight, family history, genetic makeup, the types ofpreceding or concomitant treatments, if any, and other individualcharacteristics of the subject to be treated.

“Prophylactically effective amount,” as used herein, is intended toinclude the amount of an iRNA that, when administered to a subjecthaving a disorder of lipid metabolism, is sufficient to prevent orameliorate the disease or one or more symptoms of the disease.Ameliorating the disease includes slowing the course of the disease orreducing the severity of later-developing disease. The “prophylacticallyeffective amount” may vary depending on the iRNA, how the agent isadministered, the degree of risk of disease, and the history, age,weight, family history, genetic makeup, the types of preceding orconcomitant treatments, if any, and other individual characteristics ofthe patient to be treated.

A “therapeutically-effective amount” or “prophylacticaly effectiveamount” also includes an amount of an RNAi agent that produces somedesired local or systemic effect at a reasonable benefit/risk ratioapplicable to any treatment. iRNA employed in the methods of the presentinvention may be administered in a sufficient amount to produce areasonable benefit/risk ratio applicable to such treatment.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human subjects and animal subjects without excessivetoxicity, irritation, allergic response, or other problem orcomplication, commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically-acceptable carrier” as used herein means apharmaceutically-acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, manufacturing aid (e.g.,lubricant, talc magnesium, calcium or zinc stearate, or steric acid), orsolvent encapsulating material, involved in carrying or transporting thesubject compound from one organ, or portion of the body, to anotherorgan, or portion of the body. Each carrier must be “acceptable” in thesense of being compatible with the other ingredients of the formulationand not injurious to the subject being treated. Some examples ofmaterials which can serve as pharmaceutically-acceptable carriersinclude: (1) sugars, such as lactose, glucose and sucrose; (2) starches,such as corn starch and potato starch; (3) cellulose, and itsderivatives, such as sodium carboxymethyl cellulose, ethyl cellulose andcellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7)lubricating agents, such as magnesium state, sodium lauryl sulfate andtalc; (8) excipients, such as cocoa butter and suppository waxes; (9)oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil,olive oil, corn oil and soybean oil; (10) glycols, such as propyleneglycol; (11) polyols, such as glycerin, sorbitol, mannitol andpolyethylene glycol; (12) esters, such as ethyl oleate and ethyllaurate; (13) agar; (14) buffering agents, such as magnesium hydroxideand aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17)isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pHbuffered solutions; (21) polyesters, polycarbonates and/orpolyanhydrides; (22) bulking agents, such as polypeptides and aminoacids (23) serum component, such as serum albumin, HDL and LDL; and (22)other non-toxic compatible substances employed in pharmaceuticalformulations.

The term “sample,” as used herein, includes a collection of similarfluids, cells, or tissues isolated from a subject, as well as fluids,cells, or tissues present within a subject. Examples of biologicalfluids include blood, serum and serosal fluids, plasma, cerebrospinalfluid, ocular fluids, lymph, urine, saliva, and the like. Tissue samplesmay include samples from tissues, organs or localized regions. Forexample, samples may be derived from particular organs, parts of organs,or fluids or cells within those organs. In certain embodiments, samplesmay be derived from the liver (e.g., whole liver or certain segments ofliver or certain types of cells in the liver, such as, e.g.,hepatocytes). In some embodiments, a “sample derived from a subject”refers to blood or plasma drawn from the subject.

II. iRNAs of the Invention

Described herein are iRNAs which inhibit the expression of an ANGPTL3gene. In one embodiment, the iRNA agent includes double-strandedribonucleic acid (dsRNA) molecules for inhibiting the expression of anANGPTL3 gene in a cell, such as a cell within a subject, e.g., a mammal,such as a human having a disorder of lipid metabolism, e.g., familialhyperlipidemia. The dsRNA includes an antisense strand having a regionof complementarity which is complementary to at least a part of an mRNAformed in the expression of an ANGPTL3gene, The region ofcomplementarity is about 30 nucleotides or less in length (e.g., about30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, or 18 nucleotides orless in length). Upon contact with a cell expressing the ANGPTL3 gene,the iRNA inhibits the expression of the ANGPTL3 gene (e.g., a human, aprimate, a non-primate, or a bird ANGPTL3 gene) by at least about 10% asassayed by, for example, a PCR or branched DNA (bDNA)-based method, orby a protein-based method, such as by immunofluorescence analysis,using, for example, Western Blotting or flowcytometric techniques.

A dsRNA includes two RNA strands that are complementary and hybridize toform a duplex structure under conditions in which the dsRNA will beused. One strand of a dsRNA (the antisense strand) includes a region ofcomplementarity that is substantially complementary, and generally fullycomplementary, to a target sequence. The target sequence can be derivedfrom the sequence of an mRNA formed during the expression of anANGPTL3gene. The other strand (the sense strand) includes a region thatis complementary to the antisense strand, such that the two strandshybridize and form a duplex structure when combined under suitableconditions. As described elsewhere herein and as known in the art, thecomplementary sequences of a dsRNA can also be contained asself-complementary regions of a single nucleic acid molecule, as opposedto being on separate oligonucleotides.

Generally, the duplex structure is between 15 and 30 base pairs inlength, e.g., between, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23,15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27,18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28,19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29,20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29,21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs in length.Ranges and lengths intermediate to the above recited ranges and lengthsare also contemplated to be part of the invention.

Similarly, the region of complementarity to the target sequence isbetween 15 and 30 nucleotides in length, e.g., between 15-29, 15-28,15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18,15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22,18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23,19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24,20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24,21-23, or 21-22 nucleotides in length. Ranges and lengths intermediateto the above recited ranges and lengths are also contemplated to be partof the invention.

In some embodiments, the dsRNA is between about 15 and about 20nucleotides in length, or between about 25 and about 30 nucleotides inlength. In general, the dsRNA is long enough to serve as a substrate forthe Dicer enzyme. For example, it is well known in the art that dsRNAslonger than about 21-23 nucleotides can serve as substrates for Dicer.As the ordinarily skilled person will also recognize, the region of anRNA targeted for cleavage will most often be part of a larger RNAmolecule, often an mRNA molecule. Where relevant, a “part” of an mRNAtarget is a contiguous sequence of an mRNA target of sufficient lengthto allow it to be a substrate for RNAi-directed cleavage (i.e., cleavagethrough a RISC pathway).

One of skill in the art will also recognize that the duplex region is aprimary functional portion of a dsRNA, e.g., a duplex region of about 9to 36 base pairs, e.g., about 10-36, 11-36, 12-36, 13-36, 14-36, 15-36,9-35, 10-35, 11-35, 12-35, 13-35, 14-35, 15-35, 9-34, 10-34, 11-34,12-34, 13-34, 14-34, 15-34, 9-33, 10-33, 11-33, 12-33, 13-33, 14-33,15-33, 9-32, 10-32, 11-32, 12-32, 13-32, 14-32, 15-32, 9-31, 10-31,11-31, 12-31, 13-32, 14-31, 15-31, 15-30, 15-29, 15-28, 15-27, 15-26,15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30,18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20,19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21,19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22,20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22base pairs. Thus, in one embodiment, to the extent that it becomesprocessed to a functional duplex, of e.g., 15-30 base pairs, thattargets a desired RNA for cleavage, an RNA molecule or complex of RNAmolecules having a duplex region greater than 30 base pairs is a dsRNA.Thus, an ordinarily skilled artisan will recognize that in oneembodiment, a miRNA is a dsRNA. In another embodiment, a dsRNA is not anaturally occurring miRNA. In another embodiment, an iRNA agent usefulto target ANGPTL3 expression is not generated in the target cell bycleavage of a larger dsRNA.

A dsRNA as described herein can further include one or moresingle-stranded nucleotide overhangs e.g., 1, 2, 3, or 4 nucleotides.dsRNAs having at least one nucleotide overhang can have unexpectedlysuperior inhibitory properties relative to their blunt-endedcounterparts. A nucleotide overhang can comprise or consist of anucleotide/nucleoside analog, including a deoxynucleotide/nucleoside.The overhang(s) can be on the sense strand, the antisense strand or anycombination thereof. Furthermore, the nucleotide(s) of an overhang canbe present on the 5′-end, 3′-end or both ends of either an antisense orsense strand of a dsRNA.

A dsRNA can be synthesized by standard methods known in the art asfurther discussed below, e.g., by use of an automated DNA synthesizer,such as are commercially available from, for example, Biosearch, AppliedBiosystems, Inc.

iRNA compounds of the invention may be prepared using a two-stepprocedure. First, the individual strands of the double-stranded RNAmolecule are prepared separately. Then, the component strands areannealed. The individual strands of the siRNA compound can be preparedusing solution-phase or solid-phase organic synthesis or both. Organicsynthesis offers the advantage that the oligonucleotide strandscomprising unnatural or modified nucleotides can be easily prepared.Single-stranded oligonucleotides of the invention can be prepared usingsolution-phase or solid-phase organic synthesis or both.

In one aspect, a dsRNA of the invention includes at least two nucleotidesequences, a sense sequence and an anti-sense sequence. The sense strandis selected from the group of sequences provided in Tables 2, 3, 7, 8, 9and 10, and the corresponding antisense strand of the sense strand isselected from the group of sequences of Tables 2, 3, 7, 8, 9 and 10. Inthis aspect, one of the two sequences is complementary to the other ofthe two sequences, with one of the sequences being substantiallycomplementary to a sequence of an mRNA generated in the expression of anANGPTL3gene. As such, in this aspect, a dsRNA will include twooligonucleotides, where one oligonucleotide is described as the sensestrand in Tables 2, 3, 7, 8, 9 and 10, and the second oligonucleotide isdescribed as the corresponding antisense strand of the sense strand inTables 2, 3, 7, 8, 9 and 10. In one embodiment, the substantiallycomplementary sequences of the dsRNA are contained on separateoligonucleotides. In another embodiment, the substantially complementarysequences of the dsRNA are contained on a single oligonucleotide.

The skilled person is well aware that dsRNAs having a duplex structureof between about 20 and 23 base pairs, e.g., 21, base pairs have beenhailed as particularly effective in inducing RNA interference (Elbashiret al., (2001) EMBO J., 20:6877-6888). However, others have found thatshorter or longer RNA duplex structures can also be effective (Chu andRana (2007) RNA 14:1714-1719; Kim et al. (2005) Nat Biotech 23:222-226).In the embodiments described above, by virtue of the nature of theoligonucleotide sequences provided in Tables 2, 3, 7, 8, 9 and 10,dsRNAs described herein can include at least one strand of a length ofminimally 21 nucleotides. It can be reasonably expected that shorterduplexes having one of the sequences of Tables 2, 3, 7, 8, 9 and 10minus only a few nucleotides on one or both ends can be similarlyeffective as compared to the dsRNAs described above. Hence, dsRNAshaving a sequence of at least 15, 16, 17, 18, 19, 20, or more contiguousnucleotides derived from one of the sequences of Tables 2, 3, 7, 8, 9and 10, and differing in their ability to inhibit the expression of anANGPTL3gene by not more than about 5, 10, 15, 20, 25, or 30% inhibitionfrom a dsRNA comprising the full sequence, are contemplated to be withinthe scope of the present invention.

In addition, the RNAs provided in Tables 2, 3, 7, 8, 9 and 10 identify asite(s) in an ANGPTL3 transcript that is susceptible to RISC-mediatedcleavage. As such, the present invention further features iRNAs thattarget within one of these sites. As used herein, an iRNA is said totarget within a particular site of an RNA transcript if the iRNApromotes cleavage of the transcript anywhere within that particularsite. Such an iRNA will generally include at least about 15 contiguousnucleotides from one of the sequences provided in Tables 2, 3, 7, 8, 9and 10 coupled to additional nucleotide sequences taken from the regioncontiguous to the selected sequence in an ANGPTL3gene.

While a target sequence is generally about 15-30 nucleotides in length,there is wide variation in the suitability of particular sequences inthis range for directing cleavage of any given target RNA. Varioussoftware packages and the guidelines set out herein provide guidance forthe identification of optimal target sequences for any given genetarget, but an empirical approach can also be taken in which a “window”or “mask” of a given size (as a non-limiting example, 21 nucleotides) isliterally or figuratively (including, e.g., in silico) placed on thetarget RNA sequence to identify sequences in the size range that canserve as target sequences. By moving the sequence “window” progressivelyone nucleotide upstream or downstream of an initial target sequencelocation, the next potential target sequence can be identified, untilthe complete set of possible sequences is identified for any giventarget size selected. This process, coupled with systematic synthesisand testing of the identified sequences (using assays as describedherein or as known in the art) to identify those sequences that performoptimally can identify those RNA sequences that, when targeted with aniRNA agent, mediate the best inhibition of target gene expression. Thus,while the sequences identified, for example, in Tables 2, 3, 7, 8, 9 and10 represent effective target sequences, it is contemplated that furtheroptimization of inhibition efficiency can be achieved by progressively“walking the window” one nucleotide upstream or downstream of the givensequences to identify sequences with equal or better inhibitioncharacteristics.

Further, it is contemplated that for any sequence identified, e.g., inTables 2, 3, 7, 8, 9 and 10, further optimization could be achieved bysystematically either adding or removing nucleotides to generate longeror shorter sequences and testing those sequences generated by walking awindow of the longer or shorter size up or down the target RNA from thatpoint. Again, coupling this approach to generating new candidate targetswith testing for effectiveness of iRNAs based on those target sequencesin an inhibition assay as known in the art and/or as described hereincan lead to further improvements in the efficiency of inhibition.Further still, such optimized sequences can be adjusted by, e.g., theintroduction of modified nucleotides as described herein or as known inthe art, addition or changes in overhang, or other modifications asknown in the art and/or discussed herein to further optimize themolecule (e.g., increasing serum stability or circulating half-life,increasing thermal stability, enhancing transmembrane delivery,targeting to a particular location or cell type, increasing interactionwith silencing pathway enzymes, increasing release from endosomes) as anexpression inhibitor.

An iRNA as described herein can contain one or more mismatches to thetarget sequence. In one embodiment, an iRNA as described herein containsno more than 3 mismatches. If the antisense strand of the iRNA containsmismatches to a target sequence, it is preferable that the area ofmismatch is not located in the center of the region of complementarity.If the antisense strand of the iRNA contains mismatches to the targetsequence, it is preferable that the mismatch be restricted to be withinthe last 5 nucleotides from either the 5′- or 3′-end of the region ofcomplementarity. For example, for a 23 nucleotide iRNA agent the strandwhich is complementary to a region of an ANGPTL3 gene, generally doesnot contain any mismatch within the central 13 nucleotides. The methodsdescribed herein or methods known in the art can be used to determinewhether an iRNA containing a mismatch to a target sequence is effectivein inhibiting the expression of an ANGPTL3 gene. Consideration of theefficacy of iRNAs with mismatches in inhibiting expression of an ANGPTL3gene is important, especially if the particular region ofcomplementarity in an ANGPTL3 gene is known to have polymorphic sequencevariation within the population.

III. Modified iRNAs of the Invention

In one embodiment, the RNA of an iRNA of the invention, e.g., a dsRNA,is chemically modified to enhance stability or other beneficialcharacteristics. The nucleic acids featured in the invention can besynthesized and/or modified by methods well established in the art, suchas those described in “Current protocols in nucleic acid chemistry,”Beaucage, S. L. et al. (Edrs.), John Wiley & Sons, Inc., New York, N.Y.,USA, which is hereby incorporated herein by reference. Modificationsinclude, for example, end modifications, e.g., 5′-end modifications(phosphorylation, conjugation, inverted linkages) or 3′-endmodifications (conjugation, DNA nucleotides, inverted linkages, etc.);base modifications, e.g., replacement with stabilizing bases,destabilizing bases, or bases that base pair with an expanded repertoireof partners, removal of bases (abasic nucleotides), or conjugated bases;sugar modifications (e.g., at the 2′-position or 4′-position) orreplacement of the sugar; and/or backbone modifications, includingmodification or replacement of the phosphodiester linkages. Specificexamples of iRNA compounds useful in the embodiments described hereininclude, but are not limited to RNAs containing modified backbones or nonatural internucleoside linkages. RNAs having modified backbonesinclude, among others, those that do not have a phosphorus atom in thebackbone. For the purposes of this specification, and as sometimesreferenced in the art, modified RNAs that do not have a phosphorus atomin their internucleoside backbone can also be considered to beoligonucleosides. In some embodiments, a modified iRNA will have aphosphorus atom in its internucleoside backbone.

Modified RNA backbones include, for example, phosphorothioates, chiralphosphorothioates, phosphorodithioates, phosphotriesters,aminoalkylphosphotriesters, methyl and other alkyl phosphonatesincluding 3′-alkylene phosphonates and chiral phosphonates,phosphinates, phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates having normal 3′-5′ linkages, 2′-5′-linked analogs ofthese, and those having inverted polarity wherein the adjacent pairs ofnucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Varioussalts, mixed salts and free acid forms are also included.

Representative U.S. patents that teach the preparation of the abovephosphorus-containing linkages include, but are not limited to, U.S.Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799;5,587,361; 5,625,050; 6,028,188; 6,124,445; 6,160,109; 6,169,170;6,172,209; 6,239,265; 6,277,603; 6,326,199; 6,346,614; 6,444,423;6,531,590; 6,534,639; 6,608,035; 6,683,167; 6,858,715; 6,867,294;6,878,805; 7,015,315; 7,041,816; 7,273,933; 7,321,029; and RE39464, theentire contents of each of which are hereby incorporated herein byreference.

Modified RNA backbones that do not include a phosphorus atom thereinhave backbones that are formed by short chain alkyl or cycloalkylinternucleoside linkages, mixed heteroatoms and alkyl or cycloalkylinternucleoside linkages, or one or more short chain heteroatomic orheterocyclic internucleoside linkages. These include those havingmorpholino linkages (formed in part from the sugar portion of anucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S and CH₂ component parts.

Representative U.S. patents that teach the preparation of the aboveoligonucleosides include, but are not limited to, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046;5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and,5,677,439, the entire contents of each of which are hereby incorporatedherein by reference.

In other embodiments, suitable RNA mimetics are contemplated for use iniRNAs, in which both the sugar and the internucleoside linkage, i.e.,the backbone, of the nucleotide units are replaced with novel groups.The base units are maintained for hybridization with an appropriatenucleic acid target compound. One such oligomeric compound, an RNAmimetic that has been shown to have excellent hybridization properties,is referred to as a peptide nucleic acid (PNA). In PNA compounds, thesugar backbone of an RNA is replaced with an amide containing backbone,in particular an aminoethylglycine backbone. The nucleobases areretained and are bound directly or indirectly to aza nitrogen atoms ofthe amide portion of the backbone. Representative U.S. patents thatteach the preparation of PNA compounds include, but are not limited to,U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, the entire contentsof each of which are hereby incorporated herein by reference. AdditionalPNA compounds suitable for use in the iRNAs of the invention aredescribed in, for example, in Nielsen et al., Science, 1991, 254,1497-1500.

Some embodiments featured in the invention include RNAs withphosphorothioate backbones and oligonucleosides with heteroatombackbones, and in particular —CH₂—NH—CH₂—, —CH₂—N(CH₃)—O—CH₂—[known as amethylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—,—CH₂—N(CH₃)—N(CH₃)—CH₂— and —N(CH₃)—CH₂—CH₂—[wherein the nativephosphodiester backbone is represented as —O—P—O—CH₂—] of theabove-referenced U.S. Pat. No. 5,489,677, and the amide backbones of theabove-referenced U.S. Pat. No. 5,602,240. In some embodiments, the RNAsfeatured herein have morpholino backbone structures of theabove-referenced U.S. Pat. No. 5,034,506.

Modified RNAs can also contain one or more substituted sugar moieties.The iRNAs, e.g., dsRNAs, featured herein can include one of thefollowing at the 2′-position: OH; F; O—, S—, or N-alkyl; O—, S—, orN-alkenyl; O—, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl,alkenyl and alkynyl can be substituted or unsubstituted C₁ to C₁₀ alkylor C₂ to C₁₀ alkenyl and alkynyl. Exemplary suitable modificationsinclude O[(CH₂)_(n)O]_(m)CH₃, O(CH₂)._(n)OCH₃, O(CH₂),NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂).CH₃)]₂, where n and mare from 1 to about 10. In other embodiments, dsRNAs include one of thefollowing at the 2′ position: C₁ to C₁₀ lower alkyl, substituted loweralkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br,CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl,heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl,an RNA cleaving group, a reporter group, an intercalator, a group forimproving the pharmacokinetic properties of an iRNA, or a group forimproving the pharmacodynamic properties of an iRNA, and othersubstituents having similar properties. In some embodiments, themodification includes a 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃, also known as2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta, 1995,78:486-504) i.e., an alkoxy-alkoxy group. Another exemplary modificationis 2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂ group, also knownas 2′-DMAOE, as described in examples herein below, and2′-dimethylaminoethoxyethoxy (also known in the art as2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e.,2′-O—CH₂—O—CH₂—N(CH₂)₂.

Other modifications include 2′-methoxy (2′-OCH₃), 2′-aminopropoxy(2′-OCH₂CH₂CH₂NH₂) and 2′-fluoro (2′-F). Similar modifications can alsobe made at other positions on the RNA of an iRNA, particularly the 3′position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linkeddsRNAs and the 5′ position of 5′ terminal nucleotide. iRNAs can alsohave sugar mimetics such as cyclobutyl moieties in place of thepentofuranosyl sugar. Representative U.S. patents that teach thepreparation of such modified sugar structures include, but are notlimited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044;5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811;5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873;5,646,265; 5,658,873; 5,670,633; and 5,700,920, certain of which arecommonly owned with the instant application. The entire contents of eachof the foregoing are hereby incorporated herein by reference.

An iRNA can also include nucleobase (often referred to in the art simplyas “base”) modifications or substitutions. As used herein, “unmodified”or “natural” nucleobases include the purine bases adenine (A) andguanine (G), and the pyrimidine bases thymine (T), cytosine (C) anduracil (U). Modified nucleobases include other synthetic and naturalnucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine,xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkylderivatives of adenine and guanine, 2-propyl and other alkyl derivativesof adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine,5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil,cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo,8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl anal other 8-substitutedadenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyland other 5-substituted uracils and cytosines, 7-methylguanine and7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-daazaadenine and 3-deazaguanine and 3-deazaadenine. Furthernucleobases include those disclosed in U.S. Pat. No. 3,687,808, thosedisclosed in Modified Nucleosides in Biochemistry, Biotechnology andMedicine, Herdewijn, P. ed. Wiley-VCH, 2008; those disclosed in TheConcise Encyclopedia Of Polymer Science And Engineering, pages 858-859,Kroschwitz, J. L, ed. John Wiley & Sons, 1990, these disclosed byEnglisch et al., (1991) Angewandte Chemie, International Edition,30:613, and those disclosed by Sanghvi, Y S., Chapter 15, dsRNA Researchand Applications, pages 289-302, Crooke, S. T. and Lebleu, B., Ed., CRCPress, 1993. Certain of these nucleobases are particularly useful forincreasing the binding affinity of the oligomeric compounds featured inthe invention. These include 5-substituted pyrimidines, 6-azapyrimidinesand N-2, N-6 and 0-6 substituted purines, including2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.5-methylcytosine substitutions have been shown to increase nucleic acidduplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. andLebleu, B., Eds., dsRNA Research and Applications, CRC Press, BocaRaton, 1993, pp. 276-278) and are exemplary base substitutions, evenmore particularly when combined with 2′-O-methoxyethyl sugarmodifications.

Representative U.S. patents that teach the preparation of certain of theabove noted modified nucleobases as well as other modified nucleobasesinclude, but are not limited to, the above noted U.S. Pat. Nos.3,687,808, 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066;5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711;5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,681,941;5,750,692; 6,015,886; 6,147,200; 6,166,197; 6,222,025; 6,235,887;6,380,368; 6,528,640; 6,639,062; 6,617,438; 7,045,610; 7,427,672; and7,495,088, the entire contents of each of which are hereby incorporatedherein by reference.

The RNA of an iRNA can also be modified to include one or more lockednucleic acids (LNA). A locked nucleic acid is a nucleotide having amodified ribose moiety in which the ribose moiety comprises an extrabridge connecting the 2′ and 4′ carbons. This structure effectively“locks” the ribose in the 3′-endo structural conformation. The additionof locked nucleic acids to siRNAs has been shown to increase siRNAstability in serum, and to reduce off-target effects (Elmen, J. et al.,(2005) Nucleic Acids Research 33(1):439-447; Mook, O R. et al., (2007)Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic AcidsResearch 31(12):3185-3193).

Representative U.S. Patents that teach the preparation of locked nucleicacid nucleotides include, but are not limited to, the following: U.S.Pat. Nos. 6,268,490; 6,670,461; 6,794,499; 6,998,484; 7,053,207;7,084,125; and 7,399,845, the entire contents of each of which arehereby incorporated herein by reference.

Potentially stabilizing modifications to the ends of RNA molecules caninclude N-(acetylaminocaproyl)-4-hydroxyprolinol (Hyp-C6-NHAc),N-(caproyl-4-hydroxyprolinol (Hyp-C6), N-(acetyl-4-hydroxyprolinol(Hyp-NHAc), thymidine-2′-0-deoxythymidine (ether),N-(aminocaproyl)-4-hydroxyprolinol (Hyp-C6-amino),2-docosanoyl-uridine-3″-phosphate, inverted base dT(idT) and others.Disclosure of this modification can be found in PCT Publication No. WO2011/005861.

IV. iRNAs Conjugated to Ligands

Another modification of the RNA of an iRNA of the invention involveschemically linking to the RNA one or more ligands, moieties orconjugates that enhance the activity, cellular distribution or cellularuptake of the iRNA. Such moieties include but are not limited to lipidmoieties such as a cholesterol moiety (Letsinger et al., (1989) Proc.Natl. Acid. Sci. USA, 86: 6553-6556), cholic acid (Manoharan et al.,(1994) Biorg. Med. Chem. Let., 4:1053-1060), a thioether, e.g.,beryl-S-tritylthiol (Manoharan et al., (1992) Ann. N.Y. Acad. Sci.,660:306-309; Manoharan et al., (1993) Biorg. Med. Chem. Let.,3:2765-2770), a thiocholesterol (Oberhauser et al., (1992) Nucl. AcidsRes., 20:533-538), an aliphatic chain, e.g., dodecandiol or undecylresidues (Saison-Behmoaras et al., (1991) EMBO J, 10:1111-1118; Kabanovet al., (1990) FEBS Lett., 259:327-330; Svinarchuk et al., (1993)Biochimie, 75:49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol ortriethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-phosphonate(Manoharan et al., (1995) Tetrahedron Lett., 36:3651-3654; Shea et al.,(1990) Nucl. Acids Res., 18:3777-3783), a polyamine or a polyethyleneglycol chain (Manoharan et al., (1995) Nucleosides & Nucleotides,14:969-973), or adamantane acetic acid (Manoharan et al., (1995)Tetrahedron Lett., 36:3651-3654), a palmityl moiety (Mishra et al.,(1995) Biochim. Biophys. Acta, 1264:229-237), or an octadecylamine orhexylamino-carbonyloxycholesterol moiety (Crooke et al., (1996) J.Pharmacol. Exp. Ther., 277:923-937).

In one embodiment, a ligand alters the distribution, targeting orlifetime of an iRNA agent into which it is incorporated. In preferredembodiments a ligand provides an enhanced affinity for a selectedtarget, e.g., molecule, cell or cell type, compartment, e.g., a cellularor organ compartment, tissue, organ or region of the body, as, e.g.,compared to a species absent such a ligand. Preferred ligands will nottake part in duplex pairing in a duplexed nucleic acid.

Ligands can include a naturally occurring substance, such as a protein(e.g., human serum albumin (HSA), low-density lipoprotein (LDL), orglobulin); carbohydrate (e.g., a dextran, pullulan, chitin, chitosan,inulin, cyclodextrin, N-acetylglucosamine, N-acetylgalactosamine orhyaluronic acid); or a lipid. The ligand can also be a recombinant orsynthetic molecule, such as a synthetic polymer, e.g., a syntheticpolyamino acid. Examples of polyamino acids include polyamino acid is apolylysine (PLL), poly L-aspartic acid, poly L-glutamic acid,styrene-maleic acid anhydride copolymer, poly(L-lactide-co-glycolied)copolymer, divinyl ether-maleic anhydride copolymer,N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol(PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacryllicacid), N-isopropylacrylamide polymers, or polyphosphazine. Example ofpolyamines include: polyethylenimine, polylysine (PLL), spermine,spermidine, polyamine, pseudopeptide-polyamine, peptidomimeticpolyamine, dendrimer polyamine, arginine, amidine, protamine, cationiclipid, cationic porphyrin, quaternary salt of a polyamine, or an alphahelical peptide.

Ligands can also include targeting groups, e.g., a cell or tissuetargeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g.,an antibody, that binds to a specified cell type such as a kidney cell.A targeting group can be a thyrotropin, melanotropin, lectin,glycoprotein, surfactant protein A, Mucin carbohydrate, multivalentlactose, multivalent galactose, N-acetyl-galactosamine,N-acetyl-gulucosamine multivalent mannose, multivalent fucose,glycosylated polyaminoacids, multivalent galactose, transferrin,bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, asteroid, bile acid, folate, vitamin B12, vitamin A, biotin, or an RGDpeptide or RGD peptide mimetic.

Other examples of ligands include dyes, intercalating agents (e.g.acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins(TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g.,phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA),lipophilic molecules, e.g., cholesterol, cholic acid, adamantane aceticacid, 1-pyrene butyric acid, dihydrotestosterone,1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol,borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid,myristic acid,O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid,dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g.,antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino,mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]₂, polyamino, alkyl,substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin),transport/absorption facilitators (e.g., aspirin, vitamin E, folicacid), synthetic ribonucleases (e.g., imidazole, bisimidazole,histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP.

Ligands can be proteins, e.g., glycoproteins, or peptides, e.g.,molecules having a specific affinity for a co-ligand, or antibodiese.g., an antibody, that binds to a specified cell type such as a hepaticcell. Ligands can also include hormones and hormone receptors. They canalso include non-peptidic species, such as lipids, lectins,carbohydrates, vitamins, cofactors, multivalent lactose, multivalentgalactose, N-acetyl-galactosamine, N-acetyl-gulucosamine multivalentmannose, or multivalent fucose. The ligand can be, for example, alipopolysaccharide, an activator of p38 MAP kinase, or an activator ofNF-κB.

The ligand can be a substance, e.g., a drug, which can increase theuptake of the iRNA agent into the cell, for example, by disrupting thecell's cytoskeleton, e.g., by disrupting the cell's microtubules,microfilaments, and/or intermediate filaments. The drug can be, forexample, taxon, vincristine, vinblastine, cytochalasin, nocodazole,japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, ormyoservin.

In some embodiments, a ligand attached to an iRNA as described hereinacts as a pharmacokinetic modulator (PK modulator). PK modulatorsinclude lipophiles, bile acids, steroids, phospholipid analogues,peptides, protein binding agents, PEG, vitamins etc. Exemplary PKmodulators include, but are not limited to, cholesterol, fatty acids,cholic acid, lithocholic acid, dialkylglycerides, diacylglyceride,phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotinetc. Oligonucleotides that comprise a number of phosphorothioatelinkages are also known to bind to serum protein, thus shortoligonucleotides, e.g., oligonucleotides of about 5 bases, 10 bases, 15bases or 20 bases, comprising multiple of phosphorothioate linkages inthe backbone are also amenable to the present invention as ligands (e.g.as PK modulating ligands). In addition, aptamers that bind serumcomponents (e.g. serum proteins) are also suitable for use as PKmodulating ligands in the embodiments described herein.

Ligand-conjugated oligonucleotides of the invention may be synthesizedby the use of an oligonucleotide that bears a pendant reactivefunctionality, such as that derived from the attachment of a linkingmolecule onto the oligonucleotide (described below). This reactiveoligonucleotide may be reacted directly with commercially-availableligands, ligands that are synthesized bearing any of a variety ofprotecting groups, or ligands that have a linking moiety attachedthereto.

The oligonucleotides used in the conjugates of the present invention maybe conveniently and routinely made through the well-known technique ofsolid-phase synthesis. Equipment for such synthesis is sold by severalvendors including, for example, Applied Biosystems (Foster City,Calif.). Any other means for such synthesis known in the art mayadditionally or alternatively be employed. It is also known to usesimilar techniques to prepare other oligonucleotides, such as thephosphorothioates and alkylated derivatives.

In the ligand-conjugated oligonucleotides and ligand-molecule bearingsequence-specific linked nucleosides of the present invention, theoligonucleotides and oligonucleosides may be assembled on a suitable DNAsynthesizer utilizing standard nucleotide or nucleoside precursors, ornucleotide or nucleoside conjugate precursors that already bear thelinking moiety, ligand-nucleotide or nucleoside-conjugate precursorsthat already bear the ligand molecule, or non-nucleoside ligand-bearingbuilding blocks.

When using nucleotide-conjugate precursors that already bear a linkingmoiety, the synthesis of the sequence-specific linked nucleosides istypically completed, and the ligand molecule is then reacted with thelinking moiety to form the ligand-conjugated oligonucleotide. In someembodiments, the oligonucleotides or linked nucleosides of the presentinvention are synthesized by an automated synthesizer usingphosphoramidites derived from ligand-nucleoside conjugates in additionto the standard phosphoramidites and non-standard phosphoramidites thatare commercially available and routinely used in oligonucleotidesynthesis.

A. Lipid Conujugates

In one embodiment, the ligand or conjugate is a lipid or lipid-basedmolecule. Such a lipid or lipid-based molecule preferably binds a serumprotein, e.g., human serum albumin (HSA). An HSA binding ligand allowsfor distribution of the conjugate to a target tissue, e.g., a non-kidneytarget tissue of the body. For example, the target tissue can be theliver, including parenchymal cells of the liver. Other molecules thatcan bind HSA can also be used as ligands. For example, neproxin oraspirin can be used. A lipid or lipid-based ligand can (a) increaseresistance to degradation of the conjugate, (b) increase targeting ortransport into a target cell or cell membrane, and/or (c) can be used toadjust binding to a serum protein, e.g., HSA.

A lipid based ligand can be used to inhibit, e.g., control the bindingof the conjugate to a target tissue. For example, a lipid or lipid-basedligand that binds to HSA more strongly will be less likely to betargeted to the kidney and therefore less likely to be cleared from thebody. A lipid or lipid-based ligand that binds to HSA less strongly canbe used to target the conjugate to the kidney.

In a preferred embodiment, the lipid based ligand binds HSA. Preferably,it binds HSA with a sufficient affinity such that the conjugate will bepreferably distributed to a non-kidney tissue. However, it is preferredthat the affinity not be so strong that the HSA-ligand binding cannot bereversed.

In another preferred embodiment, the lipid based ligand binds HSA weaklyor not at all, such that the conjugate will be preferably distributed tothe kidney. Other moieties that target to kidney cells can also be usedin place of or in addition to the lipid based ligand.

In another aspect, the ligand is a moiety, e.g., a vitamin, which istaken up by a target cell, e.g., a proliferating cell. These areparticularly useful for treating disorders characterized by unwantedcell proliferation, e.g., of the malignant or non-malignant type, e.g.,cancer cells. Exemplary vitamins include vitamin A, E, and K. Otherexemplary vitamins include are B vitamin, e.g., folic acid, B12,riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up bytarget cells such as liver cells. Also included are HSA and low densitylipoprotein (LDL).

B. Cell Permeation Agents

In another aspect, the ligand is a cell-permeation agent, preferably ahelical cell-permeation agent. Preferably, the agent is amphipathic. Anexemplary agent is a peptide such as tat or antennopedia. If the agentis a peptide, it can be modified, including a peptidylmimetic,invertomers, non-peptide or pseudo-peptide linkages, and use of D-aminoacids. The helical agent is preferably an alpha-helical agent, whichpreferably has a lipophilic and a lipophobic phase.

The ligand can be a peptide or peptidomimetic. A peptidomimetic (alsoreferred to herein as an oligopeptidomimetic) is a molecule capable offolding into a defined three-dimensional structure similar to a naturalpeptide. The attachment of peptide and peptidomimetics to iRNA agentscan affect pharmacokinetic distribution of the iRNA, such as byenhancing cellular recognition and absorption. The peptide orpeptidomimetic moiety can be about 5-50 amino acids long, e.g., about 5,10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.

A peptide or peptidomimetic can be, for example, a cell permeationpeptide, cationic peptide, amphipathic peptide, or hydrophobic peptide(e.g., consisting primarily of Tyr, Trp or Phe). The peptide moiety canbe a dendrimer peptide, constrained peptide or crosslinked peptide. Inanother alternative, the peptide moiety can include a hydrophobicmembrane translocation sequence (MTS). An exemplary hydrophobicMTS-containing peptide is RFGF having the amino acid sequenceAAVALLPAVLLALLAP (SEQ ID NO: 13). An RFGF analogue (e.g., amino acidsequence AALLPVLLAAP (SEQ ID NO: 10) containing a hydrophobic MTS canalso be a targeting moiety. The peptide moiety can be a “delivery”peptide, which can carry large polar molecules including peptides,oligonucleotides, and protein across cell membranes. For example,sequences from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO: 11) andthe Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO: 12)have been found to be capable of functioning as delivery peptides. Apeptide or peptidomimetic can be encoded by a random sequence of DNA,such as a peptide identified from a phage-display library, orone-bead-one-compound (OBOC) combinatorial library (Lam et al., Nature,354:82-84, 1991). Examples of a peptide or peptidomimetic tethered to adsRNA agent via an incorporated monomer unit for cell targeting purposesis an arginine-glycine-aspartic acid (RGD)-peptide, or RGD mimic. Apeptide moiety can range in length from about 5 amino acids to about 40amino acids. The peptide moieties can have a structural modification,such as to increase stability or direct conformational properties. Anyof the structural modifications described below can be utilized.

An RGD peptide for use in the compositions and methods of the inventionmay be linear or cyclic, and may be modified, e.g., glyciosylated ormethylated, to facilitate targeting to a specific tissue(s).RGD-containing peptides and peptidiomimemtics may include D-amino acids,as well as synthetic RGD mimics. In addition to RGD, one can use othermoieties that target the integrin ligand. Preferred conjugates of thisligand target PECAM-1 or VEGF.

A “cell permeation peptide” is capable of permeating a cell, e.g., amicrobial cell, such as a bacterial or fungal cell, or a mammalian cell,such as a human cell. A microbial cell-permeating peptide can be, forexample, a α-helical linear peptide (e.g., LL-37 or Ceropin P1), adisulfide bond-containing peptide (e.g., α-defensin, β-defensin orbactenecin), or a peptide containing only one or two dominating aminoacids (e.g., PR-39 or indolicidin). A cell permeation peptide can alsoinclude a nuclear localization signal (NLS). For example, a cellpermeation peptide can be a bipartite amphipathic peptide, such as MPG,which is derived from the fusion peptide domain of HIV-1 gp41 and theNLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res.31:2717-2724, 2003).

C. Carbohydrate Conjugates

In some embodiments of the compositions and methods of the invention, aniRNA oligonucleotide further comprises a carbohydrate. The carbohydrateconjugated iRNA are advantageous for the in vivo delivery of nucleicacids, as well as compositions suitable for in vivo therapeutic use, asdescribed herein. As used herein, “carbohydrate” refers to a compoundwhich is either a carbohydrate per se made up of one or moremonosaccharide units having at least 6 carbon atoms (which can belinear, branched or cyclic) with an oxygen, nitrogen or sulfur atombonded to each carbon atom; or a compound having as a part thereof acarbohydrate moiety made up of one or more monosaccharide units eachhaving at least six carbon atoms (which can be linear, branched orcyclic), with an oxygen, nitrogen or sulfur atom bonded to each carbonatom. Representative carbohydrates include the sugars (mono-, di-, tri-and oligosaccharides containing from about 4, 5, 6, 7, 8, or 9monosaccharide units), and polysaccharides such as starches, glycogen,cellulose and polysaccharide gums. Specific monosaccharides include C5and above (e.g., C5, C6, C7, or C8) sugars; di- and trisaccharidesinclude sugars having two or three monosaccharide units (e.g., C5, C6,C7, or C8).

In one embodiment, a carbohydrate conjugate for use in the compositionsand methods of the invention is a monosaccharide. In one embodiment, themonosaccharide is an N-acetylgalactosamine, such as

In another embodiment, a carbohydrate conjugate for use in thecompositions and methods of the invention is selected from the groupconsisting of:

Another representative carbohydrate conjugate for use in the embodimentsdescribed herein includes, but is not limited to,

(Formula XXIII), when one of X or Y is an oligonucleotide, the other isa hydrogen.

In some embodiments, the carbohydrate conjugate further comprises one ormore additional ligands as described above, such as, but not limited to,a PK modulator and/or a cell permeation peptide.

D. Linkers

In some embodiments, the conjugate or ligand described herein can beattached to an iRNA oligonucleotide with various linkers that can becleavable or non cleavable.

The term “linker” or “linking group” means an organic moiety thatconnects two parts of a compound, e.g., covalently attaches two parts ofa compound. Linkers typically comprise a direct bond or an atom such asoxygen or sulfur, a unit such as NR8, C(O), C(O)NH, SO, SO₂, SO₂NH or achain of atoms, such as, but not limited to, substituted orunsubstituted alkyl, substituted or unsubstituted alkenyl, substitutedor unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl,heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl,heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl,heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl,alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl,alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl,alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl,alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl,alkenylheteroarylalkenyl, alkenylheteroarylalkynyl,alkynylheteroarylalkyl, alkynylheteroarylalkenyl,alkynylheteroarylalkynyl, alkylheterocyclylalkyl,alkylheterocyclylalkenyl, alkylhererocyclylalkynyl,alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl,alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl,alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl,alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl,alkynylhereroaryl, which one or more methylenes can be interrupted orterminated by O, S, S(O), SO₂, N(R8), C(O), substituted or unsubstitutedaryl, substituted or unsubstituted heteroaryl, substituted orunsubstituted heterocyclic; where R8 is hydrogen, acyl, aliphatic orsubstituted aliphatic. In one embodiment, the linker is between about1-24 atoms, 2-24, 3-24, 4-24, 5-24, 6-24, 6-18, 7-18, 8-18 atoms, 7-17,8-17, 6-16, 7-17, or 8-16 atoms.

A cleavable linking group is one which is sufficiently stable outsidethe cell, but which upon entry into a target cell is cleaved to releasethe two parts the linker is holding together. In a preferred embodiment,the cleavable linking group is cleaved at least about 10 times, 20,times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90times or more, or at least about 100 times faster in a target cell orunder a first reference condition (which can, e.g., be selected to mimicor represent intracellular conditions) than in the blood of a subject,or under a second reference condition (which can, e.g., be selected tomimic or represent conditions found in the blood or serum).

Cleavable linking groups are susceptible to cleavage agents, e.g., pH,redox potential or the presence of degradative molecules. Generally,cleavage agents are more prevalent or found at higher levels oractivities inside cells than in serum or blood. Examples of suchdegradative agents include: redox agents which are selected forparticular substrates or which have no substrate specificity, including,e.g., oxidative or reductive enzymes or reductive agents such asmercaptans, present in cells, that can degrade a redox cleavable linkinggroup by reduction; esterases; endosomes or agents that can create anacidic environment, e.g., those that result in a pH of five or lower;enzymes that can hydrolyze or degrade an acid cleavable linking group byacting as a general acid, peptidases (which can be substrate specific),and phosphatases.

A cleavable linkage group, such as a disulfide bond can be susceptibleto pH. The pH of human serum is 7.4, while the average intracellular pHis slightly lower, ranging from about 7.1-7.3. Endosomes have a moreacidic pH, in the range of 5.5-6.0, and lysosomes have an even moreacidic pH at around 5.0. Some linkers will have a cleavable linkinggroup that is cleaved at a preferred pH, thereby releasing a cationiclipid from the ligand inside the cell, or into the desired compartmentof the cell.

A linker can include a cleavable linking group that is cleavable by aparticular enzyme. The type of cleavable linking group incorporated intoa linker can depend on the cell to be targeted. For example, aliver-targeting ligand can be linked to a cationic lipid through alinker that includes an ester group. Liver cells are rich in esterases,and therefore the linker will be cleaved more efficiently in liver cellsthan in cell types that are not esterase-rich. Other cell-types rich inesterases include cells of the lung, renal cortex, and testis.

Linkers that contain peptide bonds can be used when targeting cell typesrich in peptidases, such as liver cells and synoviocytes.

In general, the suitability of a candidate cleavable linking group canbe evaluated by testing the ability of a degradative agent (orcondition) to cleave the candidate linking group. It will also bedesirable to also test the candidate cleavable linking group for theability to resist cleavage in the blood or when in contact with othernon-target tissue. Thus, one can determine the relative susceptibilityto cleavage between a first and a second condition, where the first isselected to be indicative of cleavage in a target cell and the second isselected to be indicative of cleavage in other tissues or biologicalfluids, e.g., blood or serum. The evaluations can be carried out in cellfree systems, in cells, in cell culture, in organ or tissue culture, orin whole animals. It can be useful to make initial evaluations incell-free or culture conditions and to confirm by further evaluations inwhole animals. In preferred embodiments, useful candidate compounds arecleaved at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, orabout 100 times faster in the cell (or under in vitro conditionsselected to mimic intracellular conditions) as compared to blood orserum (or under in vitro conditions selected to mimic extracellularconditions).

i. Redox Cleavable Linking Groups

In one embodiment, a cleavable linking group is a redox cleavablelinking group that is cleaved upon reduction or oxidation. An example ofreductively cleavable linking group is a disulphide linking group(—S—S—). To determine if a candidate cleavable linking group is asuitable “reductively cleavable linking group,” or for example issuitable for use with a particular iRNA moiety and particular targetingagent one can look to methods described herein. For example, a candidatecan be evaluated by incubation with dithiothreitol (DTT), or otherreducing agent using reagents know in the art, which mimic the rate ofcleavage which would be observed in a cell, e.g., a target cell. Thecandidates can also be evaluated under conditions which are selected tomimic blood or serum conditions. In one, candidate compounds are cleavedby at most about 10% in the blood. In other embodiments, usefulcandidate compounds are degraded at least about 2, 4, 10, 20, 30, 40,50, 60, 70, 80, 90, or about 100 times faster in the cell (or under invitro conditions selected to mimic intracellular conditions) as comparedto blood (or under in vitro conditions selected to mimic extracellularconditions). The rate of cleavage of candidate compounds can bedetermined using standard enzyme kinetics assays under conditions chosento mimic intracellular media and compared to conditions chosen to mimicextracellular media.

ii. Phosphate-based cleavable linking groups In another embodiment, acleavable linker comprises a phosphate-based cleavable linking group. Aphosphate-based cleavable linking group is cleaved by agents thatdegrade or hydrolyze the phosphate group. An example of an agent thatcleaves phosphate groups in cells are enzymes such as phosphatases incells. Examples of phosphate-based linking groups are —O—P(O)(ORk)-O—,—O—P(S)(ORk)-O—, —O—P(S)(SRk)-O—, —S—P(O)(ORk)-O—, —O—P(O)(ORk)-S—,—S—P(O)(ORk)-S—, —O—P(S)(ORk)-S—, —S—P(S)(ORk)-O—, —O—P(O)(Rk)-O—,—O—P(S)(Rk)-O—, —S—P(O)(Rk)-O—, —S—P(S)(Rk)-O—, —S—P(O)(Rk)-S—,—O—P(S)(Rk)-S—. Preferred embodiments are —O—P(O)(OH)—O—,—O—P(S)(OH)—O—, —O—P(S)(SH)—O—, —S—P(O)(OH)—O—, —O—P(O)(OH)—S—,—S—P(O)(OH)—S—, —O—P(S)(OH)—S—, —S—P(S)(OH)—O—, —O—P(O)(H)—O—,—O—P(S)(H)—O—, —S—P(O)(H)—O—, —S—P(S)(H)—O—, —S—P(O)(H)—S—,—O—P(S)(H)—S—. A preferred embodiment is —O—P(O)(OH)—O—. Thesecandidates can be evaluated using methods analogous to those describedabove.

iii. Acid Cleavable Linking Groups

In another embodiment, a cleavable linker comprises an acid cleavablelinking group. An acid cleavable linking group is a linking group thatis cleaved under acidic conditions. In preferred embodiments acidcleavable linking groups are cleaved in an acidic environment with a pHof about 6.5 or lower (e.g., about 6.0, 5.75, 5.5, 5.25, 5.0, or lower),or by agents such as enzymes that can act as a general acid. In a cell,specific low pH organelles, such as endosomes and lysosomes can providea cleaving environment for acid cleavable linking groups. Examples ofacid cleavable linking groups include but are not limited to hydrazones,esters, and esters of amino acids. Acid cleavable groups can have thegeneral formula —C═NN—, C(O)O, or —OC(O). A preferred embodiment is whenthe carbon attached to the oxygen of the ester (the alkoxy group) is anaryl group, substituted alkyl group, or tertiary alkyl group such asdimethyl pentyl or t-butyl. These candidates can be evaluated usingmethods analogous to those described above.

iv. Ester-based linking groups In another embodiment, a cleavable linkercomprises an ester-based cleavable linking group. An ester-basedcleavable linking group is cleaved by enzymes such as esterases andamidases in cells. Examples of ester-based cleavable linking groupsinclude but are not limited to esters of alkylene, alkenylene andalkynylene groups. Ester cleavable linking groups have the generalformula —C(O)O—, or —OC(O)—. These candidates can be evaluated usingmethods analogous to those described above.

v. Peptide-Based Cleaving Groups

In yet another embodiment, a cleavable linker comprises a peptide-basedcleavable linking group. A peptide-based cleavable linking group iscleaved by enzymes such as peptidases and proteases in cells.Peptide-based cleavable linking groups are peptide bonds formed betweenamino acids to yield oligopeptides (e.g., dipeptides, tripeptides etc.)and polypeptides. Peptide-based cleavable groups do not include theamide group (—C(O)NH—). The amide group can be formed between anyalkylene, alkenylene or alkynelene. A peptide bond is a special type ofamide bond formed between amino acids to yield peptides and proteins.The peptide based cleavage group is generally limited to the peptidebond (i.e., the amide bond) formed between amino acids yielding peptidesand proteins and does not include the entire amide functional group.Peptide-based cleavable linking groups have the general formula—NHCHRAC(O)NHCHRBC(O)—, where RA and RB are the R groups of the twoadjacent amino acids. These candidates can be evaluated using methodsanalogous to those described above.

In one embodiment, an iRNA of the invention is conjugated to acarbohydrate through a linker. Non-limiting examples of iRNAcarbohydrate conjugates with linkers of the compositions and methods ofthe invention include but are not limited to,

when one of X or Y is an oligonucleotide, the other is a hydrogen.

In certain embodiments of the compositions and methods of the invention,a ligand is one or more GalNAc (N-acetylgalactosamine) derivativesattached through a bivalent or trivalent branched linker.

In one embodiment, a dsRNA of the invention is conjugated to a bivalentor trivalent branched linker selected from the group of structures shownin any of formula (XXXI)-(XXXIV):

wherein:q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5B and q5C represent independentlyfor each occurrence 0-20 and wherein the repeating unit can be the sameor different;P^(2A), p^(2B), p^(3A), p^(3B), p^(4A), p^(4B), p^(5A), p^(5B), p^(5C),T^(2A), T^(2B), T^(3A), T^(3B), T^(4A), T^(4B), T^(4A), T^(5B), T^(5C)are each independently for each occurrence absent, CO, NH, O, S, OC(O),NHC(O), CH₂, CH₂NH or CH₂O;Q^(2A), Q^(2B), Q^(3A), Q^(3B), Q^(4B), Q^(5A), Q^(5B), Q^(5C) areindependently for each occurrence absent, alkylene, substituted alkylenewherin one or more methylenes can be interrupted or terminated by one ormore of O, S, S(O), SO₂, N(R^(N)), C(R′)═C(R″), C≡C or C(O);R^(2A), R^(2B), R^(3A), R^(3B), R^(4A), R^(4B), R^(5A), R^(5B), R^(5C)are each independently for each occurrence absent, NH, O, S, CH₂, C(O)O,C(O)NH, NHCH(R^(a))C(O), —C(O)—CH(R^(a))—NH—, CO,

or heterocyclyl;

L^(2A), L^(2B), L^(3A), L^(3B), L^(4A), L^(4B), L^(5A)A, L^(5B) andL^(5C) represent the ligand; i.e. each independently for each occurrencea monosaccharide (such as GalNAc), disaccharide, trisaccharide,tetrasaccharide, oligosaccharide, or polysaccharide; and R^(a) is H oramino acid side chain. Trivalent conjugating GalNAc derivatives areparticularly useful for use with RNAi agents for inhibiting theexpression of a target gene, such as those of formula (XXXV):

-   -   wherein L^(5A), L^(5B) and L^(5C) represent a monosaccharide,        such as GalNAc derivative.

Examples of suitable bivalent and trivalent branched linker groupsconjugating GalNAc derivatives include, but are not limited to, thestructures recited above as formulas II_VII, XI, X, and XIII.

Representative U.S. patents that teach the preparation of RNA conjugatesinclude, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882;5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717,5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077;5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735;4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335;4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830;5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536;5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203,5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810;5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923;5,599,928 and 5,688,941; 6,294,664; 6,320,017; 6,576,752; 6,783,931;6,900,297; 7,037,646; 8,106,022, the entire contents of each of whichare hereby incorporated herein by reference.

It is not necessary for all positions in a given compound to beuniformly modified, and in fact more than one of the aforementionedmodifications can be incorporated in a single compound or even at asingle nucleoside within an iRNA. The present invention also includesiRNA compounds that are chimeric compounds.

“Chimeric” iRNA compounds or “chimeras,” in the context of thisinvention, are iRNA compounds, preferably dsRNAs, which contain two ormore chemically distinct regions, each made up of at least one monomerunit, i.e., a nucleotide in the case of a dsRNA compound. These iRNAstypically contain at least one region wherein the RNA is modified so asto confer upon the iRNA increased resistance to nuclease degradation,increased cellular uptake, and/or increased binding affinity for thetarget nucleic acid. An additional region of the iRNA can serve as asubstrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. Byway of example, RNase H is a cellular endonuclease which cleaves the RNAstrand of an RNA:DNA duplex. Activation of RNase H, therefore, resultsin cleavage of the RNA target, thereby greatly enhancing the efficiencyof iRNA inhibition of gene expression. Consequently, comparable resultscan often be obtained with shorter iRNAs when chimeric dsRNAs are used,compared to phosphorothioate deoxy dsRNAs hybridizing to the same targetregion. Cleavage of the RNA target can be routinely detected by gelelectrophoresis and, if necessary, associated nucleic acid hybridizationtechniques known in the art.

In certain instances, the RNA of an iRNA can be modified by a non-ligandgroup. A number of non-ligand molecules have been conjugated to iRNAs inorder to enhance the activity, cellular distribution or cellular uptakeof the iRNA, and procedures for performing such conjugations areavailable in the scientific literature. Such non-ligand moieties haveincluded lipid moieties, such as cholesterol (Kubo, T. et al., Biochem.Biophys. Res. Comm., 2007, 365(1):54-61; Letsinger et al., Proc. Natl.Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg.Med. Chem. Lett., 1994, 4:1053), a thioether, e.g., hexyl-S-tritylthiol(Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306; Manoharan etal., Bioorg. Med. Chem. Let., 1993, 3:2765), a thiocholesterol(Oberhauser et al., Nucl. Acids Res., 1992, 20:533), an aliphatic chain,e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J.,1991, 10:111; Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk etal., Biochimie, 1993, 75:49), a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990,18:3777), a polyamine or a polyethylene glycol chain (Manoharan et al.,Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid(Manoharan et al., Tetrahedron Lett., 1995, 36:3651), a palmityl moiety(Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or anoctadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke etal., J. Pharmacol. Exp. Ther., 1996, 277:923). Representative UnitedStates patents that teach the preparation of such RNA conjugates havebeen listed above. Typical conjugation protocols involve the synthesisof an RNAs bearing an aminolinker at one or more positions of thesequence. The amino group is then reacted with the molecule beingconjugated using appropriate coupling or activating reagents. Theconjugation reaction can be performed either with the RNA still bound tothe solid support or following cleavage of the RNA, in solution phase.Purification of the RNA conjugate by HPLC typically affords the pureconjugate.

IV. Delivery of an iRNA of the Invention

The delivery of an iRNA of the invention to a cell e.g., a cell within asubject, such as a human subject (e.g., a subject in need thereof, suchas a subject having a disorder of lipid metabolism) can be achieved in anumber of different ways. For example, delivery may be performed bycontacting a cell with an iRNA of the invention either in vitro or invivo. In vivo delivery may also be performed directly by administering acomposition comprising an iRNA, e.g., a dsRNA, to a subject.Alternatively, in vivo delivery may be performed indirectly byadministering one or more vectors that encode and direct the expressionof the iRNA. These alternatives are discussed further below.

In general, any method of delivering a nucleic acid molecule (in vitroor in vivo) can be adapted for use with an iRNA of the invention (seee.g., Akhtar S. and Julian R L., (1992) Trends Cell. Biol. 2(5):139-144and WO94/02595, which are incorporated herein by reference in theirentireties). For in vivo delivery, factors to consider in order todeliver an iRNA molecule include, for example, biological stability ofthe delivered molecule, prevention of non-specific effects, andaccumulation of the delivered molecule in the target tissue. Thenon-specific effects of an iRNA can be minimized by localadministration, for example, by direct injection or implantation into atissue or topically administering the preparation. Local administrationto a treatment site maximizes local concentration of the agent, limitsthe exposure of the agent to systemic tissues that can otherwise beharmed by the agent or that can degrade the agent, and permits a lowertotal dose of the iRNA molecule to be administered. Several studies haveshown successful knockdown of gene products when an iRNA is administeredlocally. For example, intraocular delivery of a VEGF dsRNA byintravitreal injection in cynomolgus monkeys (Tolentino, M J. et al.,(2004) Retina 24:132-138) and subretinal injections in mice (Reich, S J.et al. (2003) Mol. Vis. 9:210-216) were both shown to preventneovascularization in an experimental model of age-related maculardegeneration. In addition, direct intratumoral injection of a dsRNA inmice reduces tumor volume (Pille, J. et al. (2005) Mol. Ther.11:267-274) and can prolong survival of tumor-bearing mice (Kim, W J. etal., (2006) Mol. Ther. 14:343-350; Li, S. et al., (2007) Mol. Ther.15:515-523). RNA interference has also shown success with local deliveryto the CNS by direct injection (Dorn, G. et al., (2004) Nucleic Acids32:e49; Tan, P H. et al. (2005) Gene Ther. 12:59-66; Makimura, H. et al(2002) BMC Neurosci. 3:18; Shishkina, G T., et al. (2004) Neuroscience129:521-528; Thakker, E R., et al. (2004) Proc. Natl. Acad. Sci. U.S.A.101:17270-17275; Akaneya, Y., et al. (2005) J. Neurophysiol. 93:594-602)and to the lungs by intranasal administration (Howard, K A. et al.,(2006) Mol. Ther. 14:476-484; Zhang, X. et al., (2004) J. Biol. Chem.279:10677-10684; Bitko, V. et al., (2005) Nat. Med. 11:50-55). Foradministering an iRNA systemically for the treatment of a disease, theRNA can be modified or alternatively delivered using a drug deliverysystem; both methods act to prevent the rapid degradation of the dsRNAby endo- and exo-nucleases in vivo. Modification of the RNA or thepharmaceutical carrier can also permit targeting of the iRNA compositionto the target tissue and avoid undesirable off-target effects. iRNAmolecules can be modified by chemical conjugation to lipophilic groupssuch as cholesterol to enhance cellular uptake and prevent degradation.For example, an iRNA directed against ApoB conjugated to a lipophiliccholesterol moiety was injected systemically into mice and resulted inknockdown of apoB mRNA in both the liver and jejunum (Soutschek, J. etal., (2004) Nature 432:173-178). Conjugation of an iRNA to an aptamerhas been shown to inhibit tumor growth and mediate tumor regression in amouse model of prostate cancer (McNamara, J O. et al., (2006) Nat.Biotechnol. 24:1005-1015). In an alternative embodiment, the iRNA can bedelivered using drug delivery systems such as a nanoparticle, adendrimer, a polymer, liposomes, or a cationic delivery system.Positively charged cationic delivery systems facilitate binding of aniRNA molecule (negatively charged) and also enhance interactions at thenegatively charged cell membrane to permit efficient uptake of an iRNAby the cell. Cationic lipids, dendrimers, or polymers can either bebound to an iRNA, or induced to form a vesicle or micelle (see e.g., KimS H. et al., (2008) Journal of Controlled Release 129(2):107-116) thatencases an iRNA. The formation of vesicles or micelles further preventsdegradation of the iRNA when administered systemically. Methods formaking and administering cationic-iRNA complexes are well within theabilities of one skilled in the art (see e.g., Sorensen, D R., et al.(2003) J. Mol. Biol 327:761-766; Verma, U N. et al., (2003) Clin. CancerRes. 9:1291-1300; Arnold, A S et al., (2007) J. Hypertens. 25:197-205,which are incorporated herein by reference in their entirety). Somenon-limiting examples of drug delivery systems useful for systemicdelivery of iRNAs include DOTAP (Sorensen, D R., et al (2003), supra;Verma, U N. et al., (2003), supra), Oligofectamine, “solid nucleic acidlipid particles” (Zimmermann, T S. et al., (2006) Nature 441:111-114),cardiolipin (Chien, P Y. et al., (2005) Cancer Gene Ther. 12:321-328;Pal, A. et al., (2005) Int J. Oncol. 26:1087-1091), polyethyleneimine(Bonnet M E. et al., (2008) Pharm. Res. August 16 Epub ahead of print;Aigner, A. (2006) J. Biomed. Biotechnol. 71659), Arg-Gly-Asp (RGD)peptides (Liu, S. (2006) Mol. Pharm. 3:472-487), and polyamidoamines(Tomalia, D A. et al., (2007) Biochem. Soc. Trans. 35:61-67; Yoo, H. etal., (1999) Pharm. Res. 16:1799-1804). In some embodiments, an iRNAforms a complex with cyclodextrin for systemic administration. Methodsfor administration and pharmaceutical compositions of iRNAs andcyclodextrins can be found in U.S. Pat. No. 7,427,605, which is hereinincorporated by reference in its entirety.

A. Vector Encoded iRNAs of the Invention

iRNA targeting the ANGPTL3 gene can be expressed from transcriptionunits inserted into DNA or RNA vectors (see, e.g., Couture, A, et al.,TIG. (1996), 12:5-10; Skillern, A., et al., International PCTPublication No. WO 00/22113, Conrad, International PCT Publication No.WO 00/22114, and Conrad, U.S. Pat. No. 6,054,299). Expression can betransient (on the order of hours to weeks) or sustained (weeks to monthsor longer), depending upon the specific construct used and the targettissue or cell type. These transgenes can be introduced as a linearconstruct, a circular plasmid, or a viral vector, which can be anintegrating or non-integrating vector. The transgene can also beconstructed to permit it to be inherited as an extrachromosomal plasmid(Gassmann, et al., (1995) Proc. Natl. Acad. Sci. USA 92:1292).

The individual strand or strands of an iRNA can be transcribed from apromoter on an expression vector. Where two separate strands are to beexpressed to generate, for example, a dsRNA, two separate expressionvectors can be co-introduced (e.g., by transfection or infection) into atarget cell. Alternatively each individual strand of a dsRNA can betranscribed by promoters both of which are located on the sameexpression plasmid.

In one embodiment, a dsRNA is expressed as inverted repeatpolynucleotides joined by a linker polynucleotide sequence such that thedsRNA has a stem and loop structure. iRNA expression vectors aregenerally DNA plasmids or viral vectors. Expression vectors compatiblewith eukaryotic cells, preferably those compatible with vertebratecells, can be used to produce recombinant constructs for the expressionof an iRNA as described herein. Eukaryotic cell expression vectors arewell known in the art and are available from a number of commercialsources. Typically, such vectors are provided containing convenientrestriction sites for insertion of the desired nucleic acid segment.Delivery of iRNA expressing vectors can be systemic, such as byintravenous or intramuscular administration, by administration to targetcells ex-planted from the patient followed by reintroduction into thepatient, or by any other means that allows for introduction into adesired target cell.

iRNA expression plasmids can be transfected into target cells as acomplex with cationic lipid carriers (e.g., Oligofectamine) ornon-cationic lipid-based carriers (e.g., Transit-TKO™). Multiple lipidtransfections for iRNA-mediated knockdowns targeting different regionsof a target RNA over a period of a week or more are also contemplated bythe invention. Successful introduction of vectors into host cells can bemonitored using various known methods. For example, transienttransfection can be signaled with a reporter, such as a fluorescentmarker, such as Green Fluorescent Protein (GFP). Stable transfection ofcells ex vivo can be ensured using markers that provide the transfectedcell with resistance to specific environmental factors (e.g.,antibiotics and drugs), such as hygromycin B resistance.

Viral vector systems which can be utilized with the methods andcompositions described herein include, but are not limited to, (a)adenovirus vectors; (b) retrovirus vectors, including but not limited tolentiviral vectors, moloney murine leukemia virus, etc.; (c)adeno-associated virus vectors; (d) herpes simplex virus vectors; (e) SV40 vectors; (f) polyoma virus vectors; (g) papilloma virus vectors; (h)picornavirus vectors; (i) pox virus vectors such as an orthopox, e.g.,vaccinia virus vectors or avipox, e.g. canary pox or fowl pox; and (j) ahelper-dependent or gutless adenovirus. Replication-defective virusescan also be advantageous. Different vectors will or will not becomeincorporated into the cells' genome. The constructs can include viralsequences for transfection, if desired. Alternatively, the construct canbe incorporated into vectors capable of episomal replication, e.g. EPVand EBV vectors. Constructs for the recombinant expression of an iRNAwill generally require regulatory elements, e.g., promoters, enhancers,etc., to ensure the expression of the iRNA in target cells. Otheraspects to consider for vectors and constructs are further describedbelow.

Vectors useful for the delivery of an iRNA will include regulatoryelements (promoter, enhancer, etc.) sufficient for expression of theiRNA in the desired target cell or tissue. The regulatory elements canbe chosen to provide either constitutive or regulated/inducibleexpression.

Expression of the iRNA can be precisely regulated, for example, by usingan inducible regulatory sequence that is sensitive to certainphysiological regulators, e.g., circulating glucose levels, or hormones(Docherty et al., 1994, FASEB J. 8:20-24). Such inducible expressionsystems, suitable for the control of dsRNA expression in cells or inmammals include, for example, regulation by ecdysone, by estrogen,progesterone, tetracycline, chemical inducers of dimerization, andisopropyl-beta-D1-thiogalactopyranoside (IPTG). A person skilled in theart would be able to choose the appropriate regulatory/promoter sequencebased on the intended use of the iRNA transgene.

Viral vectors that contain nucleic acid sequences encoding an iRNA canbe used. For example, a retroviral vector can be used (see Miller etal., (1993) Meth. Enzymol. 217:581-599). These retroviral vectorscontain the components necessary for the correct packaging of the viralgenome and integration into the host cell DNA. The nucleic acidsequences encoding an iRNA are cloned into one or more vectors, whichfacilitate delivery of the nucleic acid into a patient. More detailabout retroviral vectors can be found, for example, in Boesen et al.,Biotherapy 6:291-302 (1994), which describes the use of a retroviralvector to deliver the mdr1 gene to hematopoietic stem cells in order tomake the stem cells more resistant to chemotherapy. Other referencesillustrating the use of retroviral vectors in gene therapy are: Cloweset al., (1994) J. Clin. Invest. 93:644-651; Kiem et al., (1994) Blood83:1467-1473; Salmons and Gunzberg, (1993) Human Gene Therapy 4:129-141;and Grossman and Wilson, (1993) Curr. Opin. in Genetics and Devel.3:110-114. Lentiviral vectors contemplated for use include, for example,the HIV based vectors described in U.S. Pat. Nos. 6,143,520; 5,665,557;and 5,981,276, which are herein incorporated by reference.

Adenoviruses are also contemplated for use in delivery of iRNAs of theinvention. Adenoviruses are especially attractive vehicles, e.g., fordelivering genes to respiratory epithelia. Adenoviruses naturally infectrespiratory epithelia where they cause a mild disease. Other targets foradenovirus-based delivery systems are liver, the central nervous system,endothelial cells, and muscle. Adenoviruses have the advantage of beingcapable of infecting non-dividing cells. Kozarsky and Wilson, (1993)Current Opinion in Genetics and Development 3:499-503 present a reviewof adenovirus-based gene therapy. Bout et al., (1994) Human Gene Therapy5:3-10 demonstrated the use of adenovirus vectors to transfer genes tothe respiratory epithelia of rhesus monkeys. Other instances of the useof adenoviruses in gene therapy can be found in Rosenfeld et al., (1991)Science 252:431-434; Rosenfeld et al., (1992) Cell 68:143-155;Mastrangeli et al., (1993) J. Clin. Invest. 91:225-234; PCT PublicationWO94/12649; and Wang et al., (1995) Gene Therapy 2:775-783. A suitableAV vector for expressing an iRNA featured in the invention, a method forconstructing the recombinant AV vector, and a method for delivering thevector into target cells, are described in Xia H et al. (2002), Nat.Biotech. 20: 1006-1010.

Adeno-associated virus (AAV) vectors may also be used to delivery aniRNA of the invention (Walsh et al., (1993) Proc. Soc. Exp. Biol. Med.204:289-300; U.S. Pat. No. 5,436,146). In one embodiment, the iRNA canbe expressed as two separate, complementary single-stranded RNAmolecules from a recombinant AAV vector having, for example, either theU6 or H1 RNA promoters, or the cytomegalovirus (CMV) promoter. SuitableAAV vectors for expressing the dsRNA featured in the invention, methodsfor constructing the recombinant AV vector, and methods for deliveringthe vectors into target cells are described in Samulski R et al. (1987),J. Virol. 61: 3096-3101; Fisher K J et al. (1996), J. Virol, 70:520-532; Samulski R et al. (1989), J. Virol. 63: 3822-3826; U.S. Pat.Nos. 5,252,479; 5,139,941; International Patent Application No. WO94/13788; and International Patent Application No. WO 93/24641, theentire disclosures of which are herein incorporated by reference.

Another viral vector suitable for delivery of an iRNA of the inevtion isa pox virus such as a vaccinia virus, for example an attenuated vacciniasuch as Modified Virus Ankara (MVA) or NYVAC, an avipox such as fowl poxor canary pox.

The tropism of viral vectors can be modified by pseudotyping the vectorswith envelope proteins or other surface antigens from other viruses, orby substituting different viral capsid proteins, as appropriate. Forexample, lentiviral vectors can be pseudotyped with surface proteinsfrom vesicular stomatitis virus (VSV), rabies, Ebola, Mokola, and thelike. AAV vectors can be made to target different cells by engineeringthe vectors to express different capsid protein serotypes; see, e.g.,Rabinowitz J E et al. (2002), J Virol 76:791-801, the entire disclosureof which is herein incorporated by reference.

The pharmaceutical preparation of a vector can include the vector in anacceptable diluent, or can include a slow release matrix in which thegene delivery vehicle is imbedded. Alternatively, where the completegene delivery vector can be produced intact from recombinant cells,e.g., retroviral vectors, the pharmaceutical preparation can include oneor more cells which produce the gene delivery system.

V. Pharmaceutical Compositions of the Invention

The present invention also includes pharmaceutical compositions andformulations which include the iRNAs of the invention. In oneembodiment, provided herein are pharmaceutical compositions containingan iRNA, as described herein, and a pharmaceutically acceptable carrier.The pharmaceutical compositions containing the iRNA are useful fortreating a disease or disorder associated with the expression oractivity of an ANGPTL3 gene, e.g., a disorder of lipid metabolism, suchas hypertriglyceridemia.

Such pharmaceutical compositions are formulated based on the mode ofdelivery. One example is compositions that are formulated for systemicadministration via parenteral delivery, e.g., by intravenous (IV) or forsubcutaneous delivery. Another example is compositions that areformulated for direct delivery into the liver, e.g., by infusion intothe liver, such as by continuous pump infusion.

The pharmaceutical compositions of the invention may be administered indosages sufficient to inhibit expression of a ANGPTL3 gene. In general,a suitable dose of an iRNA of the invention will be in the range ofabout 0.001 to about 200.0 milligrams per kilogram body weight of therecipient per day, generally in the range of about 1 to 50 mg perkilogram body weight per day. For example, the dsRNA can be administeredat about 0.01 mg/kg, about 0.05 mg/kg, about 0.5 mg/kg, about 1 mg/kg,about 1.5 mg/kg, about 2 mg/kg, about 3 mg/kg, about 10 mg/kg, about 20mg/kg, about 30 mg/kg, about 40 mg/kg, or about 50 mg/kg per singledose.

For example, the dsRNA may be administered at a dose of about 0.1, 0.2,0.3, 0.4, 0.5, 0.6, 0.7. 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,1.8. 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8. 2.9, 3, 3.1, 3.2,3.3, 3.4, 3.5, 3.6, 3.7, 3.8. 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7,4.8. 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8. 5.9, 6, 6.1, 6.2,6.3, 6.4, 6.5, 6.6, 6.7, 6.8. 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7,7.8. 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8. 8.9, 9, 9.1, 9.2,9.3, 9.4, 9.5, 9.6, 9.7, 9.8. 9.9, or about 10 mg/kg. Values and rangesintermediate to the recited values are also intended to be part of thisinvention.

In another embodiment, the dsRNA is administered at a dose of about 0.1to about 50 mg/kg, about 0.25 to about 50 mg/kg, about 0.5 to about 50mg/kg, about 0.75 to about 50 mg/kg, about 1 to about 50 mg/mg, about1.5 to about 50 mg/kb, about 2 to about 50 mg/kg, about 2.5 to about 50mg/kg, about 3 to about 50 mg/kg, about 3.5 to about 50 mg/kg, about 4to about 50 mg/kg, about 4.5 to about 50 mg/kg, about 5 to about 50mg/kg, about 7.5 to about 50 mg/kg, about 10 to about 50 mg/kg, about 15to about 50 mg/kg, about 20 to about 50 mg/kg, about 20 to about 50mg/kg, about 25 to about 50 mg/kg, about 25 to about 50 mg/kg, about 30to about 50 mg/kg, about 35 to about 50 mg/kg, about 40 to about 50mg/kg, about 45 to about 50 mg/kg, about 0.1 to about 45 mg/kg, about0.25 to about 45 mg/kg, about 0.5 to about 45 mg/kg, about 0.75 to about45 mg/kg, about 1 to about 45 mg/mg, about 1.5 to about 45 mg/kb, about2 to about 45 mg/kg, about 2.5 to about 45 mg/kg, about 3 to about 45mg/kg, about 3.5 to about 45 mg/kg, about 4 to about 45 mg/kg, about 4.5to about 45 mg/kg, about 5 to about 45 mg/kg, about 7.5 to about 45mg/kg, about 10 to about 45 mg/kg, about 15 to about 45 mg/kg, about 20to about 45 mg/kg, about 20 to about 45 mg/kg, about 25 to about 45mg/kg, about 25 to about 45 mg/kg, about 30 to about 45 mg/kg, about 35to about 45 mg/kg, about 40 to about 45 mg/kg, about 0.1 to about 40mg/kg, about 0.25 to about 40 mg/kg, about 0.5 to about 40 mg/kg, about0.75 to about 40 mg/kg, about 1 to about 40 mg/mg, about 1.5 to about 40mg/kb, about 2 to about 40 mg/kg, about 2.5 to about 40 mg/kg, about 3to about 40 mg/kg, about 3.5 to about 40 mg/kg, about 4 to about 40mg/kg, about 4.5 to about 40 mg/kg, about 5 to about 40 mg/kg, about 7.5to about 40 mg/kg, about 10 to about 40 mg/kg, about 15 to about 40mg/kg, about 20 to about 40 mg/kg, about 20 to about 40 mg/kg, about 25to about 40 mg/kg, about 25 to about 40 mg/kg, about 30 to about 40mg/kg, about 35 to about 40 mg/kg, about 0.1 to about 30 mg/kg, about0.25 to about 30 mg/kg, about 0.5 to about 30 mg/kg, about 0.75 to about30 mg/kg, about 1 to about 30 mg/mg, about 1.5 to about 30 mg/kb, about2 to about 30 mg/kg, about 2.5 to about 30 mg/kg, about 3 to about 30mg/kg, about 3.5 to about 30 mg/kg, about 4 to about 30 mg/kg, about 4.5to about 30 mg/kg, about 5 to about 30 mg/kg, about 7.5 to about 30mg/kg, about 10 to about 30 mg/kg, about 15 to about 30 mg/kg, about 20to about 30 mg/kg, about 20 to about 30 mg/kg, about 25 to about 30mg/kg, about 0.1 to about 20 mg/kg, about 0.25 to about 20 mg/kg, about0.5 to about 20 mg/kg, about 0.75 to about 20 mg/kg, about 1 to about 20mg/mg, about 1.5 to about 20 mg/kb, about 2 to about 20 mg/kg, about 2.5to about 20 mg/kg, about 3 to about 20 mg/kg, about 3.5 to about 20mg/kg, about 4 to about 20 mg/kg, about 4.5 to about 20 mg/kg, about 5to about 20 mg/kg, about 7.5 to about 20 mg/kg, about 10 to about 20mg/kg, or about 15 to about 20 mg/kg. Values and ranges intermediate tothe recited values are also intended to be part of this invention.

For example, the dsRNA may be administered at a dose of about 0.01,0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5,0.6, 0.7. 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8. 1.9, 2,2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8. 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5,3.6, 3.7, 3.8. 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8. 4.9, 5,5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8. 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5,6.6, 6.7, 6.8. 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8. 7.9, 8,8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8. 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5,9.6, 9.7, 9.8. 9.9, or about 10 mg/kg. Values and ranges intermediate tothe recited values are also intended to be part of this invention.

In another embodiment, the dsRNA is administered at a dose of about 0.5to about 50 mg/kg, about 0.75 to about 50 mg/kg, about 1 to about 50mg/mg, about 1.5 to about 50 mg/kb, about 2 to about 50 mg/kg, about 2.5to about 50 mg/kg, about 3 to about 50 mg/kg, about 3.5 to about 50mg/kg, about 4 to about 50 mg/kg, about 4.5 to about 50 mg/kg, about 5to about 50 mg/kg, about 7.5 to about 50 mg/kg, about 10 to about 50mg/kg, about 15 to about 50 mg/kg, about 20 to about 50 mg/kg, about 20to about 50 mg/kg, about 25 to about 50 mg/kg, about 25 to about 50mg/kg, about 30 to about 50 mg/kg, about 35 to about 50 mg/kg, about 40to about 50 mg/kg, about 45 to about 50 mg/kg, about 0.5 to about 45mg/kg, about 0.75 to about 45 mg/kg, about 1 to about 45 mg/mg, about1.5 to about 45 mg/kb, about 2 to about 45 mg/kg, about 2.5 to about 45mg/kg, about 3 to about 45 mg/kg, about 3.5 to about 45 mg/kg, about 4to about 45 mg/kg, about 4.5 to about 45 mg/kg, about 5 to about 45mg/kg, about 7.5 to about 45 mg/kg, about 10 to about 45 mg/kg, about 15to about 45 mg/kg, about 20 to about 45 mg/kg, about 20 to about 45mg/kg, about 25 to about 45 mg/kg, about 25 to about 45 mg/kg, about 30to about 45 mg/kg, about 35 to about 45 mg/kg, about 40 to about 45mg/kg, about 0.5 to about 40 mg/kg, about 0.75 to about 40 mg/kg, about1 to about 40 mg/mg, about 1.5 to about 40 mg/kb, about 2 to about 40mg/kg, about 2.5 to about 40 mg/kg, about 3 to about 40 mg/kg, about 3.5to about 40 mg/kg, about 4 to about 40 mg/kg, about 4.5 to about 40mg/kg, about 5 to about 40 mg/kg, about 7.5 to about 40 mg/kg, about 10to about 40 mg/kg, about 15 to about 40 mg/kg, about 20 to about 40mg/kg, about 20 to about 40 mg/kg, about 25 to about 40 mg/kg, about 25to about 40 mg/kg, about 30 to about 40 mg/kg, about 35 to about 40mg/kg, about 0.5 to about 30 mg/kg, about 0.75 to about 30 mg/kg, about1 to about 30 mg/mg, about 1.5 to about 30 mg/kb, about 2 to about 30mg/kg, about 2.5 to about 30 mg/kg, about 3 to about 30 mg/kg, about 3.5to about 30 mg/kg, about 4 to about 30 mg/kg, about 4.5 to about 30mg/kg, about 5 to about 30 mg/kg, about 7.5 to about 30 mg/kg, about 10to about 30 mg/kg, about 15 to about 30 mg/kg, about 20 to about 30mg/kg, about 20 to about 30 mg/kg, about 25 to about 30 mg/kg, about 0.5to about 20 mg/kg, about 0.75 to about 20 mg/kg, about 1 to about 20mg/mg, about 1.5 to about 20 mg/kb, about 2 to about 20 mg/kg, about 2.5to about 20 mg/kg, about 3 to about 20 mg/kg, about 3.5 to about 20mg/kg, about 4 to about 20 mg/kg, about 4.5 to about 20 mg/kg, about 5to about 20 mg/kg, about 7.5 to about 20 mg/kg, about 10 to about 20mg/kg, or about 15 to about 20 mg/kg. Values and ranges intermediate tothe recited values are also intended to be part of this invention.

For example, subjects can be administered a therapeutic amount of iRNA,such as about 0.5, 0.6, 0.7. 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6,1.7, 1.8. 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8. 2.9, 3, 3.1,3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8. 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6,4.7, 4.8. 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8. 5.9, 6, 6.1,6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8. 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6,7.7, 7.8. 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8. 8.9, 9, 9.1,9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8. 9.9, 10.5, 11, 11.5, 12, 12.5, 13,13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or about 50 mg/kg.Values and ranges intermediate to the recited values are also intendedto be part of this invention.

The pharmaceutical composition can be administered once daily, or theiRNA can be administered as two, three, or more sub-doses at appropriateintervals throughout the day or even using continuous infusion ordelivery through a controlled release formulation. In that case, theiRNA contained in each sub-dose must be correspondingly smaller in orderto achieve the total daily dosage. The dosage unit can also becompounded for delivery over several days, e.g., using a conventionalsustained release formulation which provides sustained release of theiRNA over a several day period. Sustained release formulations are wellknown in the art and are particularly useful for delivery of agents at aparticular site, such as could be used with the agents of the presentinvention. In this embodiment, the dosage unit contains a correspondingmultiple of the daily dose.

The effect of a single dose on ANGPTL3 levels can be long lasting, suchthat subsequent doses are administered at not more than 3, 4, or 5 dayintervals, or at not more than 1, 2, 3, or 4 week intervals.

The skilled artisan will appreciate that certain factors can influencethe dosage and timing required to effectively treat a subject, includingbut not limited to the severity of the disease or disorder, previoustreatments, the general health and/or age of the subject, and otherdiseases present. Moreover, treatment of a subject with atherapeutically effective amount of a composition can include a singletreatment or a series of treatments. Estimates of effective dosages andin vivo half-lives for the individual iRNAs encompassed by the inventioncan be made using conventional methodologies or on the basis of in vivotesting using an appropriate animal model, as described elsewhereherein.

Advances in mouse genetics have generated a number of mouse models forthe study of various human diseases, such as disorders of lipidmetabolism that would benefit from reduction in the expression ofANGPTL3. Such models can be used for in vivo testing of iRNA, as well asfor determining a therapeutically effective dose. Suitable mouse modelsare known in the art and include, for example, an obese (ob/ob) mousecontaining a mutation in the obese (ob) gene (Wiegman et al., (2003)Diabetes, 52:1081-1089); a mouse containing homozygous knock-out of anLDL receptor (LDLR −/− mouse; Ishibashi et al., (1993) J Clin Invest92(2):883-893); diet-induced artherosclerosis mouse model (Ishida etal., (1991) J. Lipid. Res., 32:559-568); and heterozygous lipoproteinlipase knockout mouse model (Weistock et al., (1995) J. Clin. Invest.96(6):2555-2568).

The pharmaceutical compositions of the present invention can beadministered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration can be topical (e.g., by a transdermal patch), pulmonary,e.g., by inhalation or insufflation of powders or aerosols, including bynebulizer; intratracheal, intranasal, epidermal and transdermal, oral orparenteral. Parenteral administration includes intravenous,intraarterial, subcutaneous, intraperitoneal or intramuscular injectionor infusion; subdermal, e.g., via an implanted device; or intracranial,e.g., by intraparenchymal, intrathecal or intraventricular,administration.

The iRNA can be delivered in a manner to target a particular tissue,such as the liver (e.g., the hepatocytes of the liver).

Pharmaceutical compositions and formulations for topical administrationcan include transdermal patches, ointments, lotions, creams, gels,drops, suppositories, sprays, liquids and powders. Conventionalpharmaceutical carriers, aqueous, powder or oily bases, thickeners andthe like can be necessary or desirable. Coated condoms, gloves and thelike can also be useful. Suitable topical formulations include those inwhich the iRNAs featured in the invention are in admixture with atopical delivery agent such as lipids, liposomes, fatty acids, fattyacid esters, steroids, chelating agents and surfactants. Suitable lipidsand liposomes include neutral (e.g., dioleoylphosphatidyl DOPEethanolamine, dimyristoylphosphatidyl choline DMPC,distearolyphosphatidyl choline) negative (e.g., dimyristoylphosphatidylglycerol DMPG) and cationic (e.g., dioleoyltetramethylaminopropyl DOTAPand dioleoylphosphatidyl ethanolamine DOTMA). iRNAs featured in theinvention can be encapsulated within liposomes or can form complexesthereto, in particular to cationic liposomes. Alternatively, iRNAs canbe complexed to lipids, in particular to cationic lipids. Suitable fattyacids and esters include but are not limited to arachidonic acid, oleicacid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristicacid, palmitic acid, stearic acid, linoleic acid, linolenic acid,dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or aC₁₋₂₀ alkyl ester (e.g., isopropylmyristate IPM), monoglyceride,diglyceride or pharmaceutically acceptable salt thereof. Topicalformulations are described in detail in U.S. Pat. No. 6,747,014, whichis incorporated herein by reference.

A. iRNA Formulations Comprising Membranous Molecular Assemblies

An iRNA for use in the compositions and methods of the invention can beformulated for delivery in a membranous molecular assembly, e.g., aliposome or a micelle. As used herein, the term “liposome” refers to avesicle composed of amphiphilic lipids arranged in at least one bilayer,e.g., one bilayer or a plurality of bilayers. Liposomes includeunilamellar and multilamellar vesicles that have a membrane formed froma lipophilic material and an aqueous interior. The aqueous portioncontains the iRNA composition. The lipophilic material isolates theaqueous interior from an aqueous exterior, which typically does notinclude the iRNA composition, although in some examples, it may.Liposomes are useful for the transfer and delivery of active ingredientsto the site of action. Because the liposomal membrane is structurallysimilar to biological membranes, when liposomes are applied to a tissue,the liposomal bilayer fuses with bilayer of the cellular membranes. Asthe merging of the liposome and cell progresses, the internal aqueouscontents that include the iRNA are delivered into the cell where theiRNA can specifically bind to a target RNA and can mediate RNAi. In somecases the liposomes are also specifically targeted, e.g., to direct theiRNA to particular cell types.

A liposome containing a RNAi agent can be prepared by a variety ofmethods. In one example, the lipid component of a liposome is dissolvedin a detergent so that micelles are formed with the lipid component. Forexample, the lipid component can be an amphipathic cationic lipid orlipid conjugate. The detergent can have a high critical micelleconcentration and may be nonionic. Exemplary detergents include cholate,CHAPS, octylglucoside, deoxycholate, and lauroyl sarcosine. The RNAiagent preparation is then added to the micelles that include the lipidcomponent. The cationic groups on the lipid interact with the RNAi agentand condense around the RNAi agent to form a liposome. Aftercondensation, the detergent is removed, e.g., by dialysis, to yield aliposomal preparation of RNAi agent.

If necessary a carrier compound that assists in condensation can beadded during the condensation reaction, e.g., by controlled addition.For example, the carrier compound can be a polymer other than a nucleicacid (e.g., spermine or spermidine). pH can also adjusted to favorcondensation.

Methods for producing stable polynucleotide delivery vehicles, whichincorporate a polynucleotide/cationic lipid complex as structuralcomponents of the delivery vehicle, are further described in, e.g., WO96/37194, the entire contents of which are incorporated herein byreference. Liposome formation can also include one or more aspects ofexemplary methods described in Felgner, P. L. et al., (1987) Proc. Natl.Acad. Sci. USA 8:7413-7417; U.S. Pat. Nos. 4,897,355; 5,171,678; Banghamet al., (1965) M. Mol. Biol. 23:238; Olson et al., (1979) Biochim.Biophys. Acta 557:9; Szoka et al., (1978) Proc. Nat. Acad. Sci. 75:4194; Mayhew et al., (1984) Biochim. Biophys. Acta 775:169; Kim et al.,(1983) Biochim. Biophys. Acta 728:339; and Fukunaga et al., (1984)Endocrinol. 115:757. Commonly used techniques for preparing lipidaggregates of appropriate size for use as delivery vehicles includesonication and freeze-thaw plus extrusion (see, e.g., Mayer et al.,(1986) Biochim. Biophys. Acta 858:161. Microfluidization can be usedwhen consistently small (50 to 200 nm) and relatively uniform aggregatesare desired (Mayhew et al., (1984) Biochim. Biophys. Acta 775:169. Thesemethods are readily adapted to packaging RNAi agent preparations intoliposomes.

Liposomes fall into two broad classes. Cationic liposomes are positivelycharged liposomes which interact with the negatively charged nucleicacid molecules to form a stable complex. The positively charged nucleicacid/liposome complex binds to the negatively charged cell surface andis internalized in an endosome. Due to the acidic pH within theendosome, the liposomes are ruptured, releasing their contents into thecell cytoplasm (Wang et al. (1987) Biochem. Biophys. Res. Commun.,147:980-985).

Liposomes, which are pH-sensitive or negatively charged, entrap nucleicacids rather than complex with them. Since both the nucleic acid and thelipid are similarly charged, repulsion rather than complex formationoccurs. Nevertheless, some nucleic acid is entrapped within the aqueousinterior of these liposomes. pH sensitive liposomes have been used todeliver nucleic acids encoding the thymidine kinase gene to cellmonolayers in culture. Expression of the exogenous gene was detected inthe target cells (Zhou et al. (1992) Journal of Controlled Release,19:269-274).

One major type of liposomal composition includes phospholipids otherthan naturally-derived phosphatidylcholine. Neutral liposomecompositions, for example, can be formed from dimyristoylphosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC).Anionic liposome compositions generally are formed from dimyristoylphosphatidylglycerol, while anionic fusogenic liposomes are formedprimarily from dioleoyl phosphatidylethanolamine (DOPE). Another type ofliposomal composition is formed from phosphatidylcholine (PC) such as,for example, soybean PC, and egg PC. Another type is formed frommixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

Examples of other methods to introduce liposomes into cells in vitro andin vivo include U.S. Pat. Nos. 5,283,185; 5,171,678; WO 94/00569; WO93/24640; WO 91/16024; Felgner, (1994) J. Biol. Chem. 269:2550; Nabel,(1993) Proc. Natl. Acad. Sci. 90:11307; Nabel, (1992) Human Gene Ther.3:649; Gershon, (1993) Biochem. 32:7143; and Strauss, (1992) EMBO J.11:417.

Non-ionic liposomal systems have also been examined to determine theirutility in the delivery of drugs to the skin, in particular systemscomprising non-ionic surfactant and cholesterol. Non-ionic liposomalformulations comprising Novasome™ I (glyceryldilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II(glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) wereused to deliver cyclosporin-A into the dermis of mouse skin. Resultsindicated that such non-ionic liposomal systems were effective infacilitating the deposition of cyclosporine A into different layers ofthe skin (Hu et al., (1994) S.T.P. Pharma. Sci., 4(6):466).

Liposomes also include “sterically stabilized” liposomes, a term which,as used herein, refers to liposomes comprising one or more specializedlipids that, when incorporated into liposomes, result in enhancedcirculation lifetimes relative to liposomes lacking such specializedlipids. Examples of sterically stabilized liposomes are those in whichpart of the vesicle-forming lipid portion of the liposome (A) comprisesone or more glycolipids, such as monosialoganglioside G_(M1), or (B) isderivatized with one or more hydrophilic polymers, such as apolyethylene glycol (PEG) moiety. While not wishing to be bound by anyparticular theory, it is thought in the art that, at least forsterically stabilized liposomes containing gangliosides, sphingomyelin,or PEG-derivatized lipids, the enhanced circulation half-life of thesesterically stabilized liposomes derives from a reduced uptake into cellsof the reticuloendothelial system (RES) (Allen et al., (1987) FEBSLetters, 223:42; Wu et al., (1993) Cancer Research, 53:3765).

Various liposomes comprising one or more glycolipids are known in theart. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., (1987), 507:64)reported the ability of monosialoganglioside G_(M1), galactocerebrosidesulfate and phosphatidylinositol to improve blood half-lives ofliposomes. These findings were expounded upon by Gabizon et al. (Proc.Natl. Acad. Sci. U.S.A., (1988), 85,:6949). U.S. Pat. No. 4,837,028 andWO 88/04924, both to Allen et al., disclose liposomes comprising (1)sphingomyelin and (2) the ganglioside G_(M1) or a galactocerebrosidesulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomescomprising sphingomyelin. Liposomes comprising1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Limet al).

In one embodiment, cationic liposomes are used. Cationic liposomespossess the advantage of being able to fuse to the cell membrane.Non-cationic liposomes, although not able to fuse as efficiently withthe plasma membrane, are taken up by macrophages in vivo and can be usedto deliver RNAi agents to macrophages.

Further advantages of liposomes include: liposomes obtained from naturalphospholipids are biocompatible and biodegradable; liposomes canincorporate a wide range of water and lipid soluble drugs; liposomes canprotect encapsulated RNAi agents in their internal compartments frommetabolism and degradation (Rosoff, in “Pharmaceutical Dosage Forms,”Lieberman, Rieger and Banker (Eds.), 1988, volume 1, p. 245). Importantconsiderations in the preparation of liposome formulations are the lipidsurface charge, vesicle size and the aqueous volume of the liposomes.

A positively charged synthetic cationic lipid,N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA)can be used to form small liposomes that interact spontaneously withnucleic acid to form lipid-nucleic acid complexes which are capable offusing with the negatively charged lipids of the cell membranes oftissue culture cells, resulting in delivery of RNAi agent (see, e.g.,Felgner, P. L. et al., (1987) Proc. Natl. Acad. Sci. USA 8:7413-7417,and U.S. Pat. No. 4,897,355 for a description of DOTMA and its use withDNA).

A DOTMA analogue, 1,2-bis(oleoyloxy)-3-(trimethylammonia)propane (DOTAP)can be used in combination with a phospholipid to form DNA-complexingvesicles. Lipofectin™ Bethesda Research Laboratories, Gaithersburg, Md.)is an effective agent for the delivery of highly anionic nucleic acidsinto living tissue culture cells that comprise positively charged DOTMAliposomes which interact spontaneously with negatively chargedpolynucleotides to form complexes. When enough positively chargedliposomes are used, the net charge on the resulting complexes is alsopositive. Positively charged complexes prepared in this wayspontaneously attach to negatively charged cell surfaces, fuse with theplasma membrane, and efficiently deliver functional nucleic acids into,for example, tissue culture cells. Another commercially availablecationic lipid, 1,2-bis(oleoyloxy)-3,3-(trimethylammonia)propane(“DOTAP”) (Boehringer Mannheim, Indianapolis, Ind.) differs from DOTMAin that the oleoyl moieties are linked by ester, rather than etherlinkages.

Other reported cationic lipid compounds include those that have beenconjugated to a variety of moieties including, for example,carboxyspermine which has been conjugated to one of two types of lipidsand includes compounds such as 5-carboxyspermylglycine dioctaoleoylamide(“DOGS”) (Transfectam™, Promega, Madison, Wis.) anddipalmitoylphosphatidylethanolamine 5-carboxyspermyl-amide (“DPPES”)(see, e.g., U.S. Pat. No. 5,171,678).

Another cationic lipid conjugate includes derivatization of the lipidwith cholesterol (“DC-Chol”) which has been formulated into liposomes incombination with DOPE (See, Gao, X. and Huang, L., (1991) Biochim.Biophys. Res. Commun. 179:280). Lipopolylysine, made by conjugatingpolylysine to DOPE, has been reported to be effective for transfectionin the presence of serum (Zhou, X. et al., (1991) Biochim. Biophys. Acta1065:8). For certain cell lines, these liposomes containing conjugatedcationic lipids, are said to exhibit lower toxicity and provide moreefficient transfection than the DOTMA-containing compositions. Othercommercially available cationic lipid products include DMRIE andDMRIE-HP (Vical, La Jolla, Calif.) and Lipofectamine (DOSPA) (LifeTechnology, Inc., Gaithersburg, Md.). Other cationic lipids suitable forthe delivery of oligonucleotides are described in WO 98/39359 and WO96/37194.

Liposomal formulations are particularly suited for topicaladministration, liposomes present several advantages over otherformulations. Such advantages include reduced side effects related tohigh systemic absorption of the administered drug, increasedaccumulation of the administered drug at the desired target, and theability to administer RNAi agent into the skin. In some implementations,liposomes are used for delivering RNAi agent to epidermal cells and alsoto enhance the penetration of RNAi agent into dermal tissues, e.g., intoskin. For example, the liposomes can be applied topically. Topicaldelivery of drugs formulated as liposomes to the skin has beendocumented (see, e.g., Weiner et al., (1992) Journal of Drug Targeting,vol. 2, 405-410 and du Plessis et al., (1992) Antiviral Research,18:259-265; Mannino, R. J. and Fould-Fogerite, S., (1998) Biotechniques6:682-690; Itani, T. et al., (1987) Gene 56:267-276; Nicolau, C. et al.(1987) Meth. Enzymol. 149:157-176; Straubinger, R. M. andPapahadjopoulos, D. (1983) Meth. Enzymol. 101:512-527; Wang, C. Y. andHuang, L., (1987) Proc. Natl. Acad. Sci. USA 84:7851-7855).

Non-ionic liposomal systems have also been examined to determine theirutility in the delivery of drugs to the skin, in particular systemscomprising non-ionic surfactant and cholesterol. Non-ionic liposomalformulations comprising Novasome I (glyceryldilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome II(glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) wereused to deliver a drug into the dermis of mouse skin. Such formulationswith RNAi agent are useful for treating a dermatological disorder.

Liposomes that include iRNA can be made highly deformable. Suchdeformability can enable the liposomes to penetrate through pore thatare smaller than the average radius of the liposome. For example,transfersomes are a type of deformable liposomes. Transferosomes can bemade by adding surface edge activators, usually surfactants, to astandard liposomal composition. Transfersomes that include RNAi agentcan be delivered, for example, subcutaneously by infection in order todeliver RNAi agent to keratinocytes in the skin. In order to crossintact mammalian skin, lipid vesicles must pass through a series of finepores, each with a diameter less than 50 nm, under the influence of asuitable transdermal gradient. In addition, due to the lipid properties,these transferosomes can be self-optimizing (adaptive to the shape ofpores, e.g., in the skin), self-repairing, and can frequently reachtheir targets without fragmenting, and often self-loading.

Other formulations amenable to the present invention are described inU.S. provisional application Ser. No. 61/018,616, filed Jan. 2, 2008;61/018,611, filed Jan. 2, 2008; 61/039,748, filed Mar. 26, 2008;61/047,087, filed Apr. 22, 2008 and 61/051,528, filed May 8, 2008. PCTapplication no PCT/US2007/080331, filed Oct. 3, 2007 also describesformulations that are amenable to the present invention.

Transfersomes are yet another type of liposomes, and are highlydeformable lipid aggregates which are attractive candidates for drugdelivery vehicles. Transfersomes can be described as lipid dropletswhich are so highly deformable that they are easily able to penetratethrough pores which are smaller than the droplet. Transfersomes areadaptable to the environment in which they are used, e.g., they areself-optimizing (adaptive to the shape of pores in the skin),self-repairing, frequently reach their targets without fragmenting, andoften self-loading. To make transfersomes it is possible to add surfaceedge-activators, usually surfactants, to a standard liposomalcomposition. Transfersomes have been used to deliver serum albumin tothe skin. The transfersome-mediated delivery of serum albumin has beenshown to be as effective as subcutaneous injection of a solutioncontaining serum albumin.

Surfactants find wide application in formulations such as emulsions(including microemulsions) and liposomes. The most common way ofclassifying and ranking the properties of the many different types ofsurfactants, both natural and synthetic, is by the use of thehydrophile/lipophile balance (HLB). The nature of the hydrophilic group(also known as the “head”) provides the most useful means forcategorizing the different surfactants used in formulations (Rieger, inPharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988,p. 285).

If the surfactant molecule is not ionized, it is classified as anonionic surfactant. Nonionic surfactants find wide application inpharmaceutical and cosmetic products and are usable over a wide range ofpH values. In general their HLB values range from 2 to about 18depending on their structure. Nonionic surfactants include nonionicesters such as ethylene glycol esters, propylene glycol esters, glycerylesters, polyglyceryl esters, sorbitan esters, sucrose esters, andethoxylated esters. Nonionic alkanolamides and ethers such as fattyalcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylatedblock polymers are also included in this class. The polyoxyethylenesurfactants are the most popular members of the nonionic surfactantclass.

If the surfactant molecule carries a negative charge when it isdissolved or dispersed in water, the surfactant is classified asanionic. Anionic surfactants include carboxylates such as soaps, acyllactylates, acyl amides of amino acids, esters of sulfuric acid such asalkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkylbenzene sulfonates, acyl isethionates, acyl taurates andsulfosuccinates, and phosphates. The most important members of theanionic surfactant class are the alkyl sulfates and the soaps.

If the surfactant molecule carries a positive charge when it isdissolved or dispersed in water, the surfactant is classified ascationic. Cationic surfactants include quaternary ammonium salts andethoxylated amines. The quaternary ammonium salts are the most usedmembers of this class.

If the surfactant molecule has the ability to carry either a positive ornegative charge, the surfactant is classified as amphoteric. Amphotericsurfactants include acrylic acid derivatives, substituted alkylamides,N-alkylbetaines and phosphatides.

The use of surfactants in drug products, formulations and in emulsionshas been reviewed (Rieger, in Pharmaceutical Dosage Forms, MarcelDekker, Inc., New York, N.Y., 1988, p. 285).

The iRNA for use in the methods of the invention can also be provided asmicellar formulations. “Micelles” are defined herein as a particulartype of molecular assembly in which amphipathic molecules are arrangedin a spherical structure such that all the hydrophobic portions of themolecules are directed inward, leaving the hydrophilic portions incontact with the surrounding aqueous phase. The converse arrangementexists if the environment is hydrophobic.

A mixed micellar formulation suitable for delivery through transdermalmembranes may be prepared by mixing an aqueous solution of the siRNAcomposition, an alkali metal C₈ to C₂₂ alkyl sulphate, and a micelleforming compounds. Exemplary micelle forming compounds include lecithin,hyaluronic acid, pharmaceutically acceptable salts of hyaluronic acid,glycolic acid, lactic acid, chamomile extract, cucumber extract, oleicacid, linoleic acid, linolenic acid, monoolein, monooleates,monolaurates, borage oil, evening of primrose oil, menthol, trihydroxyoxo cholanyl glycine and pharmaceutically acceptable salts thereof,glycerin, polyglycerin, lysine, polylysine, triolein, polyoxyethyleneethers and analogues thereof, polidocanol alkyl ethers and analoguesthereof, chenodeoxycholate, deoxycholate, and mixtures thereof. Themicelle forming compounds may be added at the same time or afteraddition of the alkali metal alkyl sulphate. Mixed micelles will formwith substantially any kind of mixing of the ingredients but vigorousmixing in order to provide smaller size micelles.

In one method a first micellar composition is prepared which containsthe siRNA composition and at least the alkali metal alkyl sulphate. Thefirst micellar composition is then mixed with at least three micelleforming compounds to form a mixed micellar composition. In anothermethod, the micellar composition is prepared by mixing the siRNAcomposition, the alkali metal alkyl sulphate and at least one of themicelle forming compounds, followed by addition of the remaining micelleforming compounds, with vigorous mixing.

Phenol and/or m-cresol may be added to the mixed micellar composition tostabilize the formulation and protect against bacterial growth.Alternatively, phenol and/or m-cresol may be added with the micelleforming ingredients. An isotonic agent such as glycerin may also beadded after formation of the mixed micellar composition.

For delivery of the micellar formulation as a spray, the formulation canbe put into an aerosol dispenser and the dispenser is charged with apropellant. The propellant, which is under pressure, is in liquid formin the dispenser. The ratios of the ingredients are adjusted so that theaqueous and propellant phases become one, i.e., there is one phase. Ifthere are two phases, it is necessary to shake the dispenser prior todispensing a portion of the contents, e.g., through a metered valve. Thedispensed dose of pharmaceutical agent is propelled from the meteredvalve in a fine spray.

Propellants may include hydrogen-containing chlorofluorocarbons,hydrogen-containing fluorocarbons, dimethyl ether and diethyl ether. Incertain embodiments, HFA 134a (1,1,1,2 tetrafluoroethane) may be used.

The specific concentrations of the essential ingredients can bedetermined by relatively straightforward experimentation. For absorptionthrough the oral cavities, it is often desirable to increase, e.g., atleast double or triple, the dosage for through injection oradministration through the gastrointestinal tract.

B. Nucleic Acid Lipid Particles

iRNAs, e.g., dsRNAs of in the invention may be fully encapsulated in thelipid formulation, e.g., to form a SPLP, pSPLP, SNALP, or other nucleicacid-lipid particle. As used herein, the term “SNALP” refers to a stablenucleic acid-lipid particle, including SPLP. As used herein, the term“SPLP” refers to a nucleic acid-lipid particle comprising plasmid DNAencapsulated within a lipid vesicle. SNALPs and SPLPs typically containa cationic lipid, a non-cationic lipid, and a lipid that preventsaggregation of the particle (e.g., a PEG-lipid conjugate). SNALPs andSPLPs are extremely useful for systemic applications, as they exhibitextended circulation lifetimes following intravenous (i.v.) injectionand accumulate at distal sites (e.g., sites physically separated fromthe administration site). SPLPs include “pSPLP,” which include anencapsulated condensing agent-nucleic acid complex as set forth in PCTPublication No. WO 00/03683. The particles of the present inventiontypically have a mean diameter of about 50 nm to about 150 nm, moretypically about 60 nm to about 130 nm, more typically about 70 nm toabout 110 nm, most typically about 70 nm to about 90 nm, and aresubstantially nontoxic. In addition, the nucleic acids when present inthe nucleic acid-lipid particles of the present invention are resistantin aqueous solution to degradation with a nuclease. Nucleic acid-lipidparticles and their method of preparation are disclosed in, e.g., U.S.Pat. Nos. 5,976,567; 5,981,501; 6,534,484; 6,586,410; 6,815,432; U.S.Publication No. 2010/0324120 and PCT Publication No. WO 96/40964.

In one embodiment, the lipid to drug ratio (mass/mass ratio) (e.g.,lipid to dsRNA ratio) will be in the range of from about 1:1 to about50:1, from about 1:1 to about 25:1, from about 3:1 to about 15:1, fromabout 4:1 to about 10:1, from about 5:1 to about 9:1, or about 6:1 toabout 9:1. Ranges intermediate to the above recited ranges are alsocontemplated to be part of the invention.

The cationic lipid can be, for example, N,N-dioleyl-N,N-dimethylammoniumchloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB),N—(I-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP),N—(I-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA),N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA),1,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA),1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA),1,2-Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP),1,2-Dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC),1,2-Dilinoleyoxy-3-morpholinopropane (DLin-MA),1,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP),1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA),1-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP),1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl),1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl),1,2-Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or3-(N,N-Dilinoleylamino)-1,2-propanediol (DLinAP),3-(N,N-Dioleylamino)-1,2-propanedio (DOAP),1,2-Dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA),1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA),2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) oranalogs thereof,(3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine(ALN100), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl4-(dimethylamino)butanoate (MC3),1,1′-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)didodecan-2-ol(Tech G1), or a mixture thereof. The cationic lipid can comprise fromabout 20 mol % to about 50 mol % or about 40 mol % of the total lipidpresent in the particle.

In another embodiment, the compound2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane can be used toprepare lipid-siRNA nanoparticles. Synthesis of2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane is described in U.S.provisional patent application No. 61/107,998 filed on Oct. 23, 2008,which is herein incorporated by reference.

In one embodiment, the lipid-siRNA particle includes 40%2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane: 10% DSPC: 40%Cholesterol: 10% PEG-C-DOMG (mole percent) with a particle size of63.0±20 nm and a 0.027 siRNA/Lipid Ratio.

The ionizable/non-cationic lipid can be an anionic lipid or a neutrallipid including, but not limited to, distearoylphosphatidylcholine(DSPC), dioleoylphosphatidylcholine (DOPC),dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol(DOPG), dipalmitoylphosphatidylglycerol (DPPG),dioleoyl-phosphatidylethanolamine (DOPE),palmitoyloleoylphosphatidylcholine (POPC),palmitoyloleoylphosphatidylethanolamine (POPE),dioleoyl-phosphatidylethanolamine4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE),distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE,16-O-dimethyl PE, 18-1-trans PE,1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), cholesterol, or amixture thereof. The non-cationic lipid can be from about 5 mol % toabout 90 mol %, about 10 mol %, or about 58 mol % if cholesterol isincluded, of the total lipid present in the particle.

The conjugated lipid that inhibits aggregation of particles can be, forexample, a polyethyleneglycol (PEG)-lipid including, without limitation,a PEG-diacylglycerol (DAG), a PEG-dialkyloxypropyl (DAA), aPEG-phospholipid, a PEG-ceramide (Cer), or a mixture thereof. ThePEG-DAA conjugate can be, for example, a PEG-dilauryloxypropyl (Ci₂), aPEG-dimyristyloxypropyl (Ci₄), a PEG-dipalmityloxypropyl (Ci₆), or aPEG-distearyloxypropyl (C]₈). The conjugated lipid that preventsaggregation of particles can be from 0 mol % to about 20 mol % or about2 mol % of the total lipid present in the particle.

In some embodiments, the nucleic acid-lipid particle further includescholesterol at, e.g., about 10 mol % to about 60 mol % or about 48 mol %of the total lipid present in the particle.

In one embodiment, the lipidoid ND98-4HC1 (MW 1487) (see U.S. patentapplication Ser. No. 12/056,230, filed Mar. 26, 2008, which isincorporated herein by reference), Cholesterol (Sigma-Aldrich), andPEG-Ceramide C16 (Avanti Polar Lipids) can be used to preparelipid-dsRNA nanoparticles (i.e., LNP01 particles). Stock solutions ofeach in ethanol can be prepared as follows: ND98, 133 mg/ml;Cholesterol, 25 mg/ml, PEG-Ceramide C16, 100 mg/ml. The ND98,Cholesterol, and PEG-Ceramide C16 stock solutions can then be combinedin a, e.g., 42:48:10 molar ratio. The combined lipid solution can bemixed with aqueous dsRNA (e.g., in sodium acetate pH 5) such that thefinal ethanol concentration is about 35-45% and the final sodium acetateconcentration is about 100-300 mM. Lipid-dsRNA nanoparticles typicallyform spontaneously upon mixing. Depending on the desired particle sizedistribution, the resultant nanoparticle mixture can be extruded througha polycarbonate membrane (e.g., 100 nm cut-off) using, for example, athermobarrel extruder, such as Lipex Extruder (Northern Lipids, Inc). Insome cases, the extrusion step can be omitted. Ethanol removal andsimultaneous buffer exchange can be accomplished by, for example,dialysis or tangential flow filtration. Buffer can be exchanged with,for example, phosphate buffered saline (PBS) at about pH 7, e.g., aboutpH 6.9, about pH 7.0, about pH 7.1, about pH 7.2, about pH 7.3, or aboutpH 7.4.

LNP01 formulations are described, e.g., in International ApplicationPublication No. WO 2008/042973, which is hereby incorporated byreference.

Additional exemplary lipid-dsRNA formulations are described in the tablebelow.

cationic lipid/non-cationic lipid/cholesterol/PEG-lipid conjugateIonizable/Cationic Lipid Lipid:siRNA ratio SNALP-11,2-Dilinolenyloxy-N,N- DLinDMA/DPPC/Cholesterol/PEG-cDMAdimethylaminopropane (DLinDMA) (57.1/7.1/34.4/1.4) lipid:siRNA ~7:12-XTC 2,2-Dilinoleyl-4- XTC/DPPC/Cholesterol/PEG-cDMAdimethylaminoethyl-[1,3]-dioxolane 57.1/7.1/34.4/1.4 (XTC) lipid:siRNA~7:1 LNP05 2,2-Dilinoleyl-4- XTC/DSPC/Cholesterol/PEG-DMGdimethylaminoethyl-[1,3]-dioxolane 57.5/7.5/31.5/3.5 (XTC) lipid:siRNA~6:1 LNP06 2,2-Dilinoleyl-4- XTC/DSPC/Cholesterol/PEG-DMGdimethylaminoethyl-[1,3]-dioxolane 57.5/7.5/31.5/3.5 (XTC) lipid:siRNA~11:1 LNP07 2,2-Dilinoleyl-4- XTC/DSPC/Cholesterol/PEG-DMGdimethylaminoethyl-[1,3]-dioxolane 60/7.5/31/1.5, (XTC) lipid:siRNA ~6:1LNP08 2,2-Dilinoleyl-4- XTC/DSPC/Cholesterol/PEG-DMGdimethylaminoethyl-[1,3]-dioxolane 60/7.5/31/1.5, (XTC) lipid:siRNA~11:1 LNP09 2,2-Dilinoleyl-4- XTC/DSPC/Cholesterol/PEG-DMGdimethylaminoethyl-[1,3]-dioxolane 50/10/38.5/1.5 (XTC) Lipid:siRNA 10:1LNP10 (3aR,5s,6aS)-N,N-dimethyl-2,2- ALN100/DSPC/Cholesterol/PEG-DMGdi((9Z,12Z)-octadeca-9,12- 50/10/38.5/1.5 dienyl)tetrahydro-3aH-Lipid:siRNA 10:1 cyclopenta[d][1,3]dioxol-5-amine (ALN100) LNP11(6Z,9Z,28Z,31Z)-heptatriaconta- MC-3/DSPC/Cholesterol/PEG-DMG6,9,28,31-tetraen-19-yl 50/10/38.5/1.5 4-(dimethylamino)butanoate (MC3)Lipid:siRNA 10:1 LNP12 1,1′-(2-(4-(2-((2-(bis(2- TechG1/DSPC/Cholesterol/PEG-DMG hydroxydodecyl)amino)ethyl)(2-50/10/38.5/1.5 hydroxydodecyl)amino)ethyl)piperazin- Lipid:siRNA 10:11-yl)ethylazanediyl)didodecan- 2-ol (Tech G1) LNP13 XTCXTC/DSPC/Chol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA: 33:1 LNP14 MC3MC3/DSPC/Chol/PEG-DMG 40/15/40/5 Lipid:siRNA: 11:1 LNP15 MC3MC3/DSPC/Chol/PEG- DSG/GalNAc-PEG-DSG 50/10/35/4.5/0.5 Lipid:siRNA: 11:1LNP16 MC3 MC3/DSPC/Chol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA: 7:1 LNP17MC3 MC3/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid:siRNA: 10:1 LNP18 MC3MC3/DSPC/Chol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA: 12:1 LNP19 MC3MC3/DSPC/Chol/PEG-DMG 50/10/35/5 Lipid:siRNA: 8:1 LNP20 MC3MC3/DSPC/Chol/PEG-DPG 50/10/38.5/1.5 Lipid:siRNA: 10:1 LNP21 C12-200C12-200/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid:siRNA: 7:1 LNP22 XTCXTC/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid:siRNA: 10:1 DSPC:distearoylphosphatidylcholine DPPC: dipalmitoylphosphatidylcholinePEG-DMG: PEG-didimyristoyl glycerol (C14-PEG, or PEG-C14) (PEG with avgmol wt of 2000) PEG-DSG: PEG-distyryl glycerol (C18-PEG, or PEG-C18)(PEG with avg mol wt of 2000) PEG-cDMA:PEG-carbamoyl-l,2-dimyristyloxypropylamine (PEG with avg mol wt of 2000)SNALP (l,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA)) comprisingformulations are described in International Publication No.W02009/127060, filed Apr. 15, 2009, which is hereby incorporated byreference. XTC comprising formulations are described, e.g., in U.S.Provisional Ser. No. 61/148,366, filed Jan. 29, 2009; U.S. ProvisionalSer. No. 61/156,851, filed Mar. 2, 2009; U.S. Provisional Ser. No. filedJun. 10, 2009; U.S. Provisional Ser. No. 61/228,373, filed Jul. 24,2009; U.S. Provisional Ser. No. 61/239,686, filed Sep. 3, 2009, andInternational Application No. PCT/US2010/022614, filed Jan. 29, 2010,which are hereby incorporated by reference. MC3 comprising formulationsare described, e.g., in U.S. Publication No. 2010/0324120, filed Jun.10, 2010, the entire contents of which are hereby incorporated byreference. ALNY-100 comprising formulations are described, e.g.,International patent application number PCT/US09/63933, filed on Nov.10, 2009, which is hereby incorporated by reference. C12-200 comprisingformulations are described in U.S. Provisional Ser. No. 61/175,770,filed May 5, 2009 and International Application No. PCT/US10/33777,filed May 5, 2010, which are hereby incorporated by reference.

Synthesis of Ionizable/Cationic Lipids

Any of the compounds, e.g., cationic lipids and the like, used in thenucleic acid-lipid particles of the invention can be prepared by knownorganic synthesis techniques, including the methods described in moredetail in the Examples. All substituents are as defined below unlessindicated otherwise.

“Alkyl” means a straight chain or branched, noncyclic or cyclic,saturated aliphatic hydrocarbon containing from 1 to 24 carbon atoms.Representative saturated straight chain alkyls include methyl, ethyl,n-propyl, n-butyl, n-pentyl, n-hexyl, and the like; while saturatedbranched alkyls include isopropyl, sec-butyl, isobutyl, tert-butyl,isopentyl, and the like. Representative saturated cyclic alkyls includecyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like; whileunsaturated cyclic alkyls include cyclopentenyl and cyclohexenyl, andthe like.

“Alkenyl” means an alkyl, as defined above, containing at least onedouble bond between adjacent carbon atoms. Alkenyls include both cis andtrans isomers. Representative straight chain and branched alkenylsinclude ethylenyl, propylenyl, 1-butenyl, 2-butenyl, isobutylenyl,1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl,2,3-dimethyl-2-butenyl, and the like.

“Alkynyl” means any alkyl or alkenyl, as defined above, whichadditionally contains at least one triple bond between adjacent carbons.Representative straight chain and branched alkynyls include acetylenyl,propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-1butynyl, and the like.

“Acyl” means any alkyl, alkenyl, or alkynyl wherein the carbon at thepoint of attachment is substituted with an oxo group, as defined below.For example, —C(═O)alkyl, —C(═O)alkenyl, and —C(═O)alkynyl are acylgroups.

“Heterocycle” means a 5- to 7-membered monocyclic, or 7- to 10-memberedbicyclic, heterocyclic ring which is either saturated, unsaturated, oraromatic, and which contains from 1 or 2 heteroatoms independentlyselected from nitrogen, oxygen and sulfur, and wherein the nitrogen andsulfur heteroatoms can be optionally oxidized, and the nitrogenheteroatom can be optionally quaternized, including bicyclic rings inwhich any of the above heterocycles are fused to a benzene ring. Theheterocycle can be attached via any heteroatom or carbon atom.Heterocycles include heteroaryls as defined below. Heterocycles includemorpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl, piperizynyl,hydantoinyl, valerolactamyl, oxiranyl, oxetanyl, tetrahydrofuranyl,tetrahydropyranyl, tetrahydropyridinyl, tetrahydroprimidinyl,tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydropyrimidinyl,tetrahydrothiophenyl, tetrahydrothiopyranyl, and the like.

The terms “optionally substituted alkyl”, “optionally substitutedalkenyl”, “optionally substituted alkynyl”, “optionally substitutedacyl”, and “optionally substituted heterocycle” means that, whensubstituted, at least one hydrogen atom is replaced with a substituent.In the case of an oxo substituent (═O) two hydrogen atoms are replaced.In this regard, substituents include oxo, halogen, heterocycle, —CN,—ORx, —NRxRy, —NRxC(═O)Ry, —NRxSO2Ry, —C(═O)Rx, —C(═O)ORx, —C(═O)NRxRy,—SOnRx and —SOnNRxRy, wherein n is 0, 1 or 2, Rx and Ry are the same ordifferent and independently hydrogen, alkyl or heterocycle, and each ofsaid alkyl and heterocycle substituents can be further substituted withone or more of oxo, halogen, —OH, —CN, alkyl, —ORx, heterocycle, —NRxRy,—NRxC(═O)Ry, —NRxSO2Ry, —C(═O)Rx, —C(═O)ORx, —C(═O)NRxR y, —SOnRx and—SOnNRxRy.

“Halogen” means fluoro, chloro, bromo and iodo.

In some embodiments, the methods of the invention can require the use ofprotecting groups. Protecting group methodology is well known to thoseskilled in the art (see, for example, Protective Groups in OrganicSynthesis, Green, T. W. et al., Wiley-Interscience, New York City,1999). Briefly, protecting groups within the context of this inventionare any group that reduces or eliminates unwanted reactivity of afunctional group. A protecting group can be added to a functional groupto mask its reactivity during certain reactions and then removed toreveal the original functional group. In some embodiments an “alcoholprotecting group” is used. An “alcohol protecting group” is any groupwhich decreases or eliminates unwanted reactivity of an alcoholfunctional group. Protecting groups can be added and removed usingtechniques well known in the art.

Synthesis of Formula A

In some embodiments, nucleic acid-lipid particles of the invention areformulated using a cationic lipid of formula A:

where R1 and R2 are independently alkyl, alkenyl or alkynyl, each can beoptionally substituted, and R3 and R4 are independently lower alkyl orR3 and R4 can be taken together to form an optionally substitutedheterocyclic ring. In some embodiments, the cationic lipid is XTC(2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane). In general, thelipid of formula A above can be made by the following Reaction Schemes 1or 2, wherein all substituents are as defined above unless indicatedotherwise.

Lipid A, where R1 and R2 are independently alkyl, alkenyl or alkynyl,each can be optionally substituted, and R3 and R4 are independentlylower alkyl or R3 and R4 can be taken together to form an optionallysubstituted heterocyclic ring, can be prepared according to Scheme 1.Ketone 1 and bromide 2 can be purchased or prepared according to methodsknown to those of ordinary skill in the art. Reaction of 1 and 2 yieldsketal 3. Treatment of ketal 3 with amine 4 yields lipids of formula A.The lipids of formula A can be converted to the corresponding ammoniumsalt with an organic salt of formula 5, where X is anion counter ionselected from halogen, hydroxide, phosphate, sulfate, or the like.

Alternatively, the ketone 1 starting material can be prepared accordingto Scheme 2. Grignard reagent 6 and cyanide 7 can be purchased orprepared according to methods known to those of ordinary skill in theart. Reaction of 6 and 7 yields ketone 1. Conversion of ketone 1 to thecorresponding lipids of formula A is as described in Scheme 1.

Synthesis of MC3

Preparation of DLin-M-C3-DMA (i.e.,(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl4-(dimethylamino)butanoate) was as follows. A solution of(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-ol (0.53 g),4-N,N-dimethylaminobutyric acid hydrochloride (0.51 g),4-N,N-dimethylaminopyridine (0.61 g) and1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.53 g) indichloromethane (5 mL) was stirred at room temperature overnight. Thesolution was washed with dilute hydrochloric acid followed by diluteaqueous sodium bicarbonate. The organic fractions were dried overanhydrous magnesium sulphate, filtered and the solvent removed on arotovap. The residue was passed down a silica gel column (20 g) using a1-5% methanol/dichloromethane elution gradient. Fractions containing thepurified product were combined and the solvent removed, yielding acolorless oil (0.54 g).

Synthesis of ALNY-100

Synthesis of ketal 519 [ALNY-100] was performed using the followingscheme 3:

Synthesis of 515

To a stirred suspension of LiAlH₄ (3.74 g, 0.09852 mol) in 200 mlanhydrous THF in a two neck RBF (1 L), was added a solution of 514 (10g, 0.04926 mol) in 70 mL of THF slowly at 0° C. under nitrogenatmosphere. After complete addition, reaction mixture was warmed to roomtemperature and then heated to reflux for 4 h. Progress of the reactionwas monitored by TLC. After completion of reaction (by TLC) the mixturewas cooled to 0° C. and quenched with careful addition of saturatedNa₂SO₄ solution. Reaction mixture was stirred for 4 h at roomtemperature and filtered off. Residue was washed well with THF. Thefiltrate and washings were mixed and diluted with 400 mL dioxane and 26mL conc. HCl and stirred for 20 minutes at room temperature. Thevolatilities were stripped off under vacuum to furnish the hydrochloridesalt of 515 as a white solid. Yield: 7.12 g ¹H-NMR (DMSO, 400 MHz):δ=9.34 (broad, 2H), 5.68 (s, 2H), 3.74 (m, 1H), 2.66-2.60 (m, 2H),2.50-2.45 (m, 5H).

Synthesis of 516

To a stirred solution of compound 515 in 100 mL dry DCM in a 250 mL twoneck RBF, was added NEt₃ (37.2 mL, 0.2669 mol) and cooled to 0° C. undernitrogen atmosphere. After a slow addition ofN-(benzyloxy-carbonyloxy)-succinimide (20 g, 0.08007 mol) in 50 mL dryDCM, reaction mixture was allowed to warm to room temperature. Aftercompletion of the reaction (2-3 h by TLC) mixture was washedsuccessively with 1N HCl solution (1×100 mL) and saturated NaHCO₃solution (1×50 mL). The organic layer was then dried over anhyd. Na2SO4and the solvent was evaporated to give crude material which was purifiedby silica gel column chromatography to get 516 as sticky mass. Yield: 11g (89%). ¹H-NMR (CDCl₃, 400 MHz): δ=7.36-7.27 (m, 5H), 5.69 (s, 2H),5.12 (s, 2H), 4.96 (br., 1H) 2.74 (s, 3H), 2.60 (m, 2H), 2.30-2.25 (m,2H). LC-MS [M+H] −232.3 (96.94%).

Synthesis of 517A and 517B

The cyclopentene 516 (5 g, 0.02164 mol) was dissolved in a solution of220 mL acetone and water (10:1) in a single neck 500 mL RBF and to itwas added N-methyl morpholine-N-oxide (7.6 g, 0.06492 mol) followed by4.2 mL of 7.6% solution of OsO4 (0.275 g, 0.00108 mol) in tert-butanolat room temperature. After completion of the reaction (˜3 h), themixture was quenched with addition of solid Na₂SO₃ and resulting mixturewas stirred for 1.5 h at room temperature. Reaction mixture was dilutedwith DCM (300 mL) and washed with water (2×100 mL) followed by saturatedNaHCO₃ (1×50 mL) solution, water (1×30 mL) and finally with brine (1×50mL). Organic phase was dried over an.Na₂SO₄ and solvent was removed invacuum. Silica gel column chromatographic purification of the crudematerial was afforded a mixture of diastereomers, which were separatedby prep HPLC. Yield: −6 g crude

517A—Peak-1 (white solid), 5.13 g (96%). 1H-NMR (DMSO, 400 MHz):δ=7.39-7.31 (m, 5H), 5.04 (s, 2H), 4.78-4.73 (m, 1H), 4.48-4.47 (d, 2H),3.94-3.93 (m, 2H), 2.71 (s, 3H), 1.72-1.67 (m, 4H). LC-MS—[M+H]-266.3,[M+NH4+]−283.5 present, HPLC-97.86%. Stereochemistry confirmed by X-ray.

Synthesis of 518

Using a procedure analogous to that described for the synthesis ofcompound 505, compound 518 (1.2 g, 41%) was obtained as a colorless oil.1H-NMR (CDCl₃, 400 MHz): δ=7.35-7.33 (m, 4H), 7.30-7.27 (m, 1H),5.37-5.27 (m, 8H), 5.12 (s, 2H), 4.75 (m, 1H), 4.58-4.57 (m, 2H),2.78-2.74 (m, 7H), 2.06-2.00 (m, 8H), 1.96-1.91 (m, 2H), 1.62 (m, 4H),1.48 (m, 2H), 1.37-1.25 (br m, 36H), 0.87 (m, 6H). HPLC-98.65%.

General Procedure for the Synthesis of Compound 519

A solution of compound 518 (1 eq) in hexane (15 mL) was added in adrop-wise fashion to an ice-cold solution of LAH in THF (1 M, 2 eq).After complete addition, the mixture was heated at 40° C. over 0.5 hthen cooled again on an ice bath. The mixture was carefully hydrolyzedwith saturated aqueous Na₂SO₄ then filtered through celite and reducedto an oil. Column chromatography provided the pure 519 (1.3 g, 68%)which was obtained as a colorless oil. ¹³C NMR δ=130.2, 130.1 (×2),127.9 (×3), 112.3, 79.3, 64.4, 44.7, 38.3, 35.4, 31.5, 29.9 (×2), 29.7,29.6 (×2), 29.5 (×3), 29.3 (×2), 27.2 (×3), 25.6, 24.5, 23.3, 226, 14.1;Electrospray MS (+ve): Molecular weight for C₄₄H₈₀NO₂ (M+H)+Calc. 654.6,Found 654.6.

Formulations prepared by either the standard or extrusion-free methodcan be characterized in similar manners. For example, formulations aretypically characterized by visual inspection. They should be whitishtranslucent solutions free from aggregates or sediment. Particle sizeand particle size distribution of lipid-nanoparticles can be measured bylight scattering using, for example, a Malvern Zetasizer Nano ZS(Malvern, USA). Particles should be about 20-300 nm, such as 40-100 nmin size. The particle size distribution should be unimodal. The totaldsRNA concentration in the formulation, as well as the entrappedfraction, is estimated using a dye exclusion assay. A sample of theformulated dsRNA can be incubated with an RNA-binding dye, such asRibogreen (Molecular Probes) in the presence or absence of a formulationdisrupting surfactant, e.g., 0.5% Triton-X100. The total dsRNA in theformulation can be determined by the signal from the sample containingthe surfactant, relative to a standard curve. The entrapped fraction isdetermined by subtracting the “free” dsRNA content (as measured by thesignal in the absence of surfactant) from the total dsRNA content.Percent entrapped dsRNA is typically >85%. For SNALP formulation, theparticle size is at least 30 nm, at least 40 nm, at least 50 nm, atleast 60 nm, at least 70 nm, at least 80 nm, at least 90 nm, at least100 nm, at least 110 nm, and at least 120 nm. The suitable range istypically about at least 50 nm to about at least 110 nm, about at least60 nm to about at least 100 nm, or about at least 80 nm to about atleast 90 nm.

Compositions and formulations for oral administration include powders orgranules, microparticulates, nanoparticulates, suspensions or solutionsin water or non-aqueous media, capsules, gel capsules, sachets, tabletsor minitablets. Thickeners, flavoring agents, diluents, emulsifiers,dispersing aids or binders can be desirable. In some embodiments, oralformulations are those in which dsRNAs featured in the invention areadministered in conjunction with one or more penetration enhancersurfactants and chelators. Suitable surfactants include fatty acidsand/or esters or salts thereof, bile acids and/or salts thereof.Suitable bile acids/salts include chenodeoxycholic acid (CDCA) andursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid,deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid,taurocholic acid, taurodeoxycholic acid, sodiumtauro-24,25-dihydro-fusidate and sodium glycodihydrofusidate. Suitablefatty acids include arachidonic acid, undecanoic acid, oleic acid,lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid,stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate,monoolein, dilaurin, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or amonoglyceride, a diglyceride or a pharmaceutically acceptable saltthereof (e.g., sodium). In some embodiments, combinations of penetrationenhancers are used, for example, fatty acids/salts in combination withbile acids/salts. One exemplary combination is the sodium salt of lauricacid, capric acid and UDCA. Further penetration enhancers includepolyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. DsRNAsfeatured in the invention can be delivered orally, in granular formincluding sprayed dried particles, or complexed to form micro ornanoparticles. DsRNA complexing agents include poly-amino acids;polyimines; polyacrylates; polyalkylacrylates, polyoxethanes,polyalkylcyanoacrylates; cationized gelatins, albumins, starches,acrylates, polyethyleneglycols (PEG) and starches;polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans,celluloses and starches. Suitable complexing agents include chitosan,N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine,polyspermines, protamine, polyvinylpyridine,polythiodiethylaminomethylethylene P(TDAE), polyaminostyrene (e.g.,p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate),poly(butylcyanoacrylate), poly(isobutylcyanoacrylate),poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate,DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate,polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolicacid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulationsfor dsRNAs and their preparation are described in detail in U.S. Pat.No. 6,887,906, US Publn. No. 20030027780, and U.S. Pat. No. 6,747,014,each of which is incorporated herein by reference.

Compositions and formulations for parenteral, intraparenchymal (into thebrain), intrathecal, intraventricular or intrahepatic administration caninclude sterile aqueous solutions which can also contain buffers,diluents and other suitable additives such as, but not limited to,penetration enhancers, carrier compounds and other pharmaceuticallyacceptable carriers or excipients.

Pharmaceutical compositions of the present invention include, but arenot limited to, solutions, emulsions, and liposome-containingformulations. These compositions can be generated from a variety ofcomponents that include, but are not limited to, preformed liquids,self-emulsifying solids and self-emulsifying semisolids. Particularlypreferred are formulations that target the liver when treating hepaticdisorders such as hepatic carcinoma.

The pharmaceutical formulations of the present invention, which canconveniently be presented in unit dosage form, can be prepared accordingto conventional techniques well known in the pharmaceutical industry.Such techniques include the step of bringing into association the activeingredients with the pharmaceutical carrier(s) or excipient(s). Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association the active ingredients with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

The compositions of the present invention can be formulated into any ofmany possible dosage forms such as, but not limited to, tablets,capsules, gel capsules, liquid syrups, soft gels, suppositories, andenemas. The compositions of the present invention can also be formulatedas suspensions in aqueous, non-aqueous or mixed media. Aqueoussuspensions can further contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension can also contain stabilizers.

C. Additional Formulations

i. Emulsions

The compositions of the present invention can be prepared and formulatedas emulsions. Emulsions are typically heterogeneous systems of oneliquid dispersed in another in the form of droplets usually exceeding0.1 m in diameter (see e.g., Ansel's Pharmaceutical Dosage Forms andDrug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004,Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199; Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., Volume 1, p. 245; Block inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335; Higuchi et al.,in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton,Pa., 1985, p. 301). Emulsions are often biphasic systems comprising twoimmiscible liquid phases intimately mixed and dispersed with each other.In general, emulsions can be of either the water-in-oil (w/o) or theoil-in-water (o/w) variety. When an aqueous phase is finely divided intoand dispersed as minute droplets into a bulk oily phase, the resultingcomposition is called a water-in-oil (w/o) emulsion. Alternatively, whenan oily phase is finely divided into and dispersed as minute dropletsinto a bulk aqueous phase, the resulting composition is called anoil-in-water (o/w) emulsion. Emulsions can contain additional componentsin addition to the dispersed phases, and the active drug which can bepresent as a solution in either aqueous phase, oily phase or itself as aseparate phase. Pharmaceutical excipients such as emulsifiers,stabilizers, dyes, and anti-oxidants can also be present in emulsions asneeded. Pharmaceutical emulsions can also be multiple emulsions that arecomprised of more than two phases such as, for example, in the case ofoil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions.Such complex formulations often provide certain advantages that simplebinary emulsions do not. Multiple emulsions in which individual oildroplets of an o/w emulsion enclose small water droplets constitute aw/o/w emulsion. Likewise a system of oil droplets enclosed in globulesof water stabilized in an oily continuous phase provides an o/w/oemulsion.

Emulsions are characterized by little or no thermodynamic stability.Often, the dispersed or discontinuous phase of the emulsion is welldispersed into the external or continuous phase and maintained in thisform through the means of emulsifiers or the viscosity of theformulation. Either of the phases of the emulsion can be a semisolid ora solid, as is the case of emulsion-style ointment bases and creams.Other means of stabilizing emulsions entail the use of emulsifiers thatcan be incorporated into either phase of the emulsion. Emulsifiers canbroadly be classified into four categories: synthetic surfactants,naturally occurring emulsifiers, absorption bases, and finely dispersedsolids (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug DeliverySystems, Allen, L V., Popovich N G., and Ansel H C., 2004, LippincottWilliams & Wilkins (8th ed.), New York, N.Y.; Idson, in PharmaceuticalDosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker,Inc., New York, N.Y., volume 1, p. 199).

Synthetic surfactants, also known as surface active agents, have foundwide applicability in the formulation of emulsions and have beenreviewed in the literature (see e.g., Ansel's Pharmaceutical DosageForms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.;Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), Marcel Dekker, Inc., New York, N.Y., 1988, volume 1, p. 199).Surfactants are typically amphiphilic and comprise a hydrophilic and ahydrophobic portion. The ratio of the hydrophilic to the hydrophobicnature of the surfactant has been termed the hydrophile/lipophilebalance (HLB) and is a valuable tool in categorizing and selectingsurfactants in the preparation of formulations. Surfactants can beclassified into different classes based on the nature of the hydrophilicgroup: nonionic, anionic, cationic and amphoteric (see e.g., Ansel'sPharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V.,Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8thed.), New York, N.Y. Rieger, in Pharmaceutical Dosage Forms, Lieberman,Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y.,volume 1, p. 285).

Naturally occurring emulsifiers used in emulsion formulations includelanolin, beeswax, phosphatides, lecithin and acacia. Absorption basespossess hydrophilic properties such that they can soak up water to formw/o emulsions yet retain their semisolid consistencies, such asanhydrous lanolin and hydrophilic petrolatum. Finely divided solids havealso been used as good emulsifiers especially in combination withsurfactants and in viscous preparations. These include polar inorganicsolids, such as heavy metal hydroxides, nonswelling clays such asbentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidalaluminum silicate and colloidal magnesium aluminum silicate, pigmentsand nonpolar solids such as carbon or glyceryl tristearate.

A large variety of non-emulsifying materials are also included inemulsion formulations and contribute to the properties of emulsions.These include fats, oils, waxes, fatty acids, fatty alcohols, fattyesters, humectants, hydrophilic colloids, preservatives and antioxidants(Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

Hydrophilic colloids or hydrocolloids include naturally occurring gumsand synthetic polymers such as polysaccharides (for example, acacia,agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth),cellulose derivatives (for example, carboxymethylcellulose andcarboxypropylcellulose), and synthetic polymers (for example, carbomers,cellulose ethers, and carboxyvinyl polymers). These disperse or swell inwater to form colloidal solutions that stabilize emulsions by formingstrong interfacial films around the dispersed-phase droplets and byincreasing the viscosity of the external phase.

Since emulsions often contain a number of ingredients such ascarbohydrates, proteins, sterols and phosphatides that can readilysupport the growth of microbes, these formulations often incorporatepreservatives. Commonly used preservatives included in emulsionformulations include methyl paraben, propyl paraben, quaternary ammoniumsalts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boricacid. Antioxidants are also commonly added to emulsion formulations toprevent deterioration of the formulation. Antioxidants used can be freeradical scavengers such as tocopherols, alkyl gallates, butylatedhydroxyanisole, butylated hydroxytoluene, or reducing agents such asascorbic acid and sodium metabisulfite, and antioxidant synergists suchas citric acid, tartaric acid, and lecithin.

The application of emulsion formulations via dermatological, oral andparenteral routes and methods for their manufacture have been reviewedin the literature (see e.g., Ansel's Pharmaceutical Dosage Forms andDrug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004,Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Emulsionformulations for oral delivery have been very widely used because ofease of formulation, as well as efficacy from an absorption andbioavailability standpoint (see e.g., Ansel's Pharmaceutical DosageForms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.;Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).Mineral-oil base laxatives, oil-soluble vitamins and high fat nutritivepreparations are among the materials that have commonly beenadministered orally as o/w emulsions.

ii. Microemulsions

In one embodiment of the present invention, the compositions of iRNAsand nucleic acids are formulated as microemulsions. A microemulsion canbe defined as a system of water, oil and amphiphile which is a singleoptically isotropic and thermodynamically stable liquid solution (seee.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems,Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams &Wilkins (8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical DosageForms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc.,New York, N.Y., volume 1, p. 245). Typically microemulsions are systemsthat are prepared by first dispersing an oil in an aqueous surfactantsolution and then adding a sufficient amount of a fourth component,generally an intermediate chain-length alcohol to form a transparentsystem. Therefore, microemulsions have also been described asthermodynamically stable, isotropically clear dispersions of twoimmiscible liquids that are stabilized by interfacial films ofsurface-active molecules (Leung and Shah, in: Controlled Release ofDrugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCHPublishers, New York, pages 185-215). Microemulsions commonly areprepared via a combination of three to five components that include oil,water, surfactant, cosurfactant and electrolyte. Whether themicroemulsion is of the water-in-oil (w/o) or an oil-in-water (o/w) typeis dependent on the properties of the oil and surfactant used and on thestructure and geometric packing of the polar heads and hydrocarbon tailsof the surfactant molecules (Schott, in Remington's PharmaceuticalSciences, Mack Publishing Co., Easton, Pa., 1985, p. 271).

The phenomenological approach utilizing phase diagrams has beenextensively studied and has yielded a comprehensive knowledge, to oneskilled in the art, of how to formulate microemulsions (see e.g.,Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins(8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 245; Block, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 335). Compared to conventional emulsions,microemulsions offer the advantage of solubilizing water-insoluble drugsin a formulation of thermodynamically stable droplets that are formedspontaneously.

Surfactants used in the preparation of microemulsions include, but arenot limited to, ionic surfactants, non-ionic surfactants, Brij 96,polyoxyethylene oleyl ethers, polyglycerol fatty acid esters,tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310),hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500),decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750),decaglycerol sequioleate (SO750), decaglycerol decaoleate (DAO750),alone or in combination with cosurfactants. The cosurfactant, usually ashort-chain alcohol such as ethanol, 1-propanol, and 1-butanol, servesto increase the interfacial fluidity by penetrating into the surfactantfilm and consequently creating a disordered film because of the voidspace generated among surfactant molecules. Microemulsions can, however,be prepared without the use of cosurfactants and alcohol-freeself-emulsifying microemulsion systems are known in the art. The aqueousphase can typically be, but is not limited to, water, an aqueoussolution of the drug, glycerol, PEG300, PEG400, polyglycerols, propyleneglycols, and derivatives of ethylene glycol. The oil phase can include,but is not limited to, materials such as Captex 300, Captex 355, CapmulMCM, fatty acid esters, medium chain (C8-C12) mono, di, andtri-glycerides, polyoxyethylated glyceryl fatty acid esters, fattyalcohols, polyglycolized glycerides, saturated polyglycolized C8-C10glycerides, vegetable oils and silicone oil.

Microemulsions are particularly of interest from the standpoint of drugsolubilization and the enhanced absorption of drugs. Lipid basedmicroemulsions (both o/w and w/o) have been proposed to enhance the oralbioavailability of drugs, including peptides (see e.g., U.S. Pat. Nos.6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al.,Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp.Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages ofimproved drug solubilization, protection of drug from enzymatichydrolysis, possible enhancement of drug absorption due tosurfactant-induced alterations in membrane fluidity and permeability,ease of preparation, ease of oral administration over solid dosageforms, improved clinical potency, and decreased toxicity (see e.g., U.S.Pat. Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides etal., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm. Sci.,1996, 85, 138-143). Often microemulsions can form spontaneously whentheir components are brought together at ambient temperature. This canbe particularly advantageous when formulating thermolabile drugs,peptides or iRNAs. Microemulsions have also been effective in thetransdermal delivery of active components in both cosmetic andpharmaceutical applications. It is expected that the microemulsioncompositions and formulations of the present invention will facilitatethe increased systemic absorption of iRNAs and nucleic acids from thegastrointestinal tract, as well as improve the local cellular uptake ofiRNAs and nucleic acids.

Microemulsions of the present invention can also contain additionalcomponents and additives such as sorbitan monostearate (Grill 3),Labrasol, and penetration enhancers to improve the properties of theformulation and to enhance the absorption of the iRNAs and nucleic acidsof the present invention. Penetration enhancers used in themicroemulsions of the present invention can be classified as belongingto one of five broad categories—surfactants, fatty acids, bile salts,chelating agents, and non-chelating non-surfactants (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Eachof these classes has been discussed above.

iii. Microparticles

an RNAi agent of the invention may be incorporated into a particle,e.g., a microparticle. Microparticles can be produced by spray-drying,but may also be produced by other methods including lyophilization,evaporation, fluid bed drying, vacuum drying, or a combination of thesetechniques.

iv. Penetration Enhancers

In one embodiment, the present invention employs various penetrationenhancers to effect the efficient delivery of nucleic acids,particularly iRNAs, to the skin of animals. Most drugs are present insolution in both ionized and nonionized forms. However, usually onlylipid soluble or lipophilic drugs readily cross cell membranes. It hasbeen discovered that even non-lipophilic drugs can cross cell membranesif the membrane to be crossed is treated with a penetration enhancer. Inaddition to aiding the diffusion of non-lipophilic drugs across cellmembranes, penetration enhancers also enhance the permeability oflipophilic drugs.

Penetration enhancers can be classified as belonging to one of fivebroad categories, i.e., surfactants, fatty acids, bile salts, chelatingagents, and non-chelating non-surfactants (see e.g., Malmsten, M.Surfactants and polymers in drug delivery, Informa Health Care, NewYork, N.Y., 2002; Lee et al., Critical Reviews in Therapeutic DrugCarrier Systems, 1991, p. 92). Each of the above mentioned classes ofpenetration enhancers are described below in greater detail.

Surfactants (or “surface-active agents”) are chemical entities which,when dissolved in an aqueous solution, reduce the surface tension of thesolution or the interfacial tension between the aqueous solution andanother liquid, with the result that absorption of iRNAs through themucosa is enhanced. In addition to bile salts and fatty acids, thesepenetration enhancers include, for example, sodium lauryl sulfate,polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether) (seee.g., Malmsten, M. Surfactants and polymers in drug delivery, InformaHealth Care, New York, N.Y., 2002; Lee et al., Critical Reviews inTherapeutic Drug Carrier Systems, 1991, p. 92); and perfluorochemicalemulsions, such as FC-43. Takahashi et al., J. Pharm. Pharmacol., 1988,40, 252).

Various fatty acids and their derivatives which act as penetrationenhancers include, for example, oleic acid, lauric acid, capric acid(n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleicacid, linolenic acid, dicaprate, tricaprate, monoolein(1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid,glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines,acylcholines, C₁₋₂₀ alkyl esters thereof (e.g., methyl, isopropyl andt-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate,caprate, myristate, palmitate, stearate, linoleate, etc.) (see e.g.,Touitou, E., et al. Enhancement in Drug Delivery, CRC Press, Danvers,Mass., 2006; Lee et al., Critical Reviews in Therapeutic Drug CarrierSystems, 1991, p. 92; Muranishi, Critical Reviews in Therapeutic DrugCarrier Systems, 1990, 7, 1-33; El Hariri et al., J. Pharm. Pharmacol.,1992, 44, 651-654).

The physiological role of bile includes the facilitation of dispersionand absorption of lipids and fat-soluble vitamins (see e.g., Malmsten,M. Surfactants and polymers in drug delivery, Informa Health Care, NewYork, N.Y., 2002; Brunton, Chapter 38 in: Goodman & Gilman's ThePharmacological Basis of Therapeutics, 9th Ed., Hardman et al. Eds.,McGraw-Hill, New York, 1996, pp. 934-935). Various natural bile salts,and their synthetic derivatives, act as penetration enhancers. Thus theterm “bile salts” includes any of the naturally occurring components ofbile as well as any of their synthetic derivatives. Suitable bile saltsinclude, for example, cholic acid (or its pharmaceutically acceptablesodium salt, sodium cholate), dehydrocholic acid (sodiumdehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid(sodium glucholate), glycholic acid (sodium glycocholate),glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid(sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate),chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid(UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodiumglycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (see e.g.,Malmsten, M. Surfactants and polymers in drug delivery, Informa HealthCare, New York, N.Y., 2002; Lee et al., Critical Reviews in TherapeuticDrug Carrier Systems, 1991, page 92; Swinyard, Chapter 39 In:Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., MackPublishing Co., Easton, Pa., 1990, pages 782-783; Muranishi, CriticalReviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Yamamoto etal., J. Pharm. Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm.Sci., 1990, 79, 579-583).

Chelating agents, as used in connection with the present invention, canbe defined as compounds that remove metallic ions from solution byforming complexes therewith, with the result that absorption of iRNAsthrough the mucosa is enhanced. With regards to their use as penetrationenhancers in the present invention, chelating agents have the addedadvantage of also serving as DNase inhibitors, as most characterized DNAnucleases require a divalent metal ion for catalysis and are thusinhibited by chelating agents (Jarrett, J. Chromatogr., 1993, 618,315-339). Suitable chelating agents include but are not limited todisodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates(e.g., sodium salicylate, 5-methoxysalicylate and homovanilate), N-acylderivatives of collagen, laureth-9 and N-amino acyl derivatives ofbeta-diketones (enamines)(see e.g., Katdare, A. et al., Excipientdevelopment for pharmaceutical, biotechnology, and drug delivery, CRCPress, Danvers, Mass., 2006; Lee et al., Critical Reviews in TherapeuticDrug Carrier Systems, 1991, page 92; Muranishi, Critical Reviews inTherapeutic Drug Carrier Systems, 1990, 7, 1-33; Buur et al., J. ControlRel., 1990, 14, 43-51).

As used herein, non-chelating non-surfactant penetration enhancingcompounds can be defined as compounds that demonstrate insignificantactivity as chelating agents or as surfactants but that nonethelessenhance absorption of iRNAs through the alimentary mucosa (see e.g.,Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990,7, 1-33). This class of penetration enhancers includes, for example,unsaturated cyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanonederivatives (Lee et al., Critical Reviews in Therapeutic Drug CarrierSystems, 1991, page 92); and non-steroidal anti-inflammatory agents suchas diclofenac sodium, indomethacin and phenylbutazone (Yamashita et al.,J. Pharm. Pharmacol., 1987, 39, 621-626).

Agents that enhance uptake of iRNAs at the cellular level can also beadded to the pharmaceutical and other compositions of the presentinvention. For example, cationic lipids, such as lipofectin (Junichi etal, U.S. Pat. No. 5,705,188), cationic glycerol derivatives, andpolycationic molecules, such as polylysine (Lollo et al., PCTApplication WO 97/30731), are also known to enhance the cellular uptakeof dsRNAs. Examples of commercially available transfection reagentsinclude, for example Lipofectamine™ (Invitrogen; Carlsbad, Calif.),Lipofectamine 2000™ (Invitrogen; Carlsbad, Calif.), 293fectin™(Invitrogen; Carlsbad, Calif.), Cellfectin™ (Invitrogen; Carlsbad,Calif.), DMRIE-C™ (Invitrogen; Carlsbad, Calif.), FreeStyle™ MAX(Invitrogen; Carlsbad, Calif.), Lipofectamine™ 2000 CD (Invitrogen;Carlsbad, Calif.), Lipofectamine™ (Invitrogen; Carlsbad, Calif.),RNAiMAX (Invitrogen; Carlsbad, Calif.), Oligofectamine™ (Invitrogen;Carlsbad, Calif.), Optifect™ (Invitrogen; Carlsbad, Calif.), X-tremeGENEQ2 Transfection Reagent (Roche; Grenzacherstrasse, Switzerland), DOTAPLiposomal Transfection Reagent (Grenzacherstrasse, Switzerland), DOSPERLiposomal Transfection Reagent (Grenzacherstrasse, Switzerland), orFugene (Grenzacherstrasse, Switzerland), Transfectam® Reagent (Promega;Madison, Wis.), TransFast™ Transfection Reagent (Promega; Madison,Wis.), Tfx™-20 Reagent (Promega; Madison, Wis.), Tfx™-50 Reagent(Promega; Madison, Wis.), DreamFect™ (OZ Biosciences; Marseille,France), EcoTransfect (OZ Biosciences; Marseille, France), TransPass^(a)D1 Transfection Reagent (New England Biolabs; Ipswich, Mass., USA),LyoVec™/LipoGen™ (Invitrogen; San Diego, Calif., USA), PerFectinTransfection Reagent (Genlantis; San Diego, Calif., USA), NeuroPORTERTransfection Reagent (Genlantis; San Diego, Calif., USA), GenePORTERTransfection reagent (Genlantis; San Diego, Calif., USA), GenePORTER 2Transfection reagent (Genlantis; San Diego, Calif., USA), CytofectinTransfection Reagent (Genlantis; San Diego, Calif., USA), BaculoPORTERTransfection Reagent (Genlantis; San Diego, Calif., USA), TroganPORTER™transfection Reagent (Genlantis; San Diego, Calif., USA), RiboFect(Bioline; Taunton, Mass., USA), PlasFect (Bioline; Taunton, Mass., USA),UniFECTOR (B-Bridge International; Mountain View, Calif., USA),SureFECTOR (B-Bridge International; Mountain View, Calif., USA), orHiFect™ (B-Bridge International, Mountain View, Calif., USA), amongothers.

Other agents can be utilized to enhance the penetration of theadministered nucleic acids, including glycols such as ethylene glycoland propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenessuch as limonene and menthone.

v. Carriers

Certain compositions of the present invention also incorporate carriercompounds in the formulation. As used herein, “carrier compound” or“carrier” can refer to a nucleic acid, or analog thereof, which is inert(i.e., does not possess biological activity per se) but is recognized asa nucleic acid by in vivo processes that reduce the bioavailability of anucleic acid having biological activity by, for example, degrading thebiologically active nucleic acid or promoting its removal fromcirculation. The coadministration of a nucleic acid and a carriercompound, typically with an excess of the latter substance, can resultin a substantial reduction of the amount of nucleic acid recovered inthe liver, kidney or other extracirculatory reservoirs, presumably dueto competition between the carrier compound and the nucleic acid for acommon receptor. For example, the recovery of a partiallyphosphorothioate dsRNA in hepatic tissue can be reduced when it iscoadministered with polyinosinic acid, dextran sulfate, polycytidic acidor 4-acetamido-4′isothiocyano-stilbene-2,2′-disulfonic acid (Miyao etal., DsRNA Res. Dev., 1995, 5, 115-121; Takakura et al., DsRNA & Nucl.Acid Drug Dev., 1996, 6, 177-183.

vi. Excipients

In contrast to a carrier compound, a “pharmaceutical carrier” or“excipient” is a pharmaceutically acceptable solvent, suspending agentor any other pharmacologically inert vehicle for delivering one or morenucleic acids to an animal. The excipient can be liquid or solid and isselected, with the planned manner of administration in mind, so as toprovide for the desired bulk, consistency, etc., when combined with anucleic acid and the other components of a given pharmaceuticalcomposition. Typical pharmaceutical carriers include, but are notlimited to, binding agents (e.g., pregelatinized maize starch,polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers(e.g., lactose and other sugars, microcrystalline cellulose, pectin,gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calciumhydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc,silica, colloidal silicon dioxide, stearic acid, metallic stearates,hydrogenated vegetable oils, corn starch, polyethylene glycols, sodiumbenzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodiumstarch glycolate, etc.); and wetting agents (e.g., sodium laurylsulphate, etc).

Pharmaceutically acceptable organic or inorganic excipients suitable fornon-parenteral administration which do not deleteriously react withnucleic acids can also be used to formulate the compositions of thepresent invention. Suitable pharmaceutically acceptable carriersinclude, but are not limited to, water, salt solutions, alcohols,polyethylene glycols, gelatin, lactose, amylose, magnesium stearate,talc, silicic acid, viscous paraffin, hydroxymethylcellulose,polyvinylpyrrolidone and the like.

Formulations for topical administration of nucleic acids can includesterile and non-sterile aqueous solutions, non-aqueous solutions incommon solvents such as alcohols, or solutions of the nucleic acids inliquid or solid oil bases. The solutions can also contain buffers,diluents and other suitable additives. Pharmaceutically acceptableorganic or inorganic excipients suitable for non-parenteraladministration which do not deleteriously react with nucleic acids canbe used.

Suitable pharmaceutically acceptable excipients include, but are notlimited to, water, salt solutions, alcohol, polyethylene glycols,gelatin, lactose, amylose, magnesium stearate, talc, silicic acid,viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and thelike.

vii. Other Components

The compositions of the present invention can additionally contain otheradjunct components conventionally found in pharmaceutical compositions,at their art-established usage levels. Thus, for example, thecompositions can contain additional, compatible, pharmaceutically-activematerials such as, for example, antipruritics, astringents, localanesthetics or anti-inflammatory agents, or can contain additionalmaterials useful in physically formulating various dosage forms of thecompositions of the present invention, such as dyes, flavoring agents,preservatives, antioxidants, opacifiers, thickening agents andstabilizers. However, such materials, when added, should not undulyinterfere with the biological activities of the components of thecompositions of the present invention. The formulations can besterilized and, if desired, mixed with auxiliary agents, e.g.,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, colorings, flavoringsand/or aromatic substances and the like which do not deleteriouslyinteract with the nucleic acid(s) of the formulation.

Aqueous suspensions can contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran.

The suspension can also contain stabilizers.

In some embodiments, pharmaceutical compositions featured in theinvention include (a) one or more iRNA compounds and (b) one or moreagents which function by a non-RNAi mechanism and which are useful intreating a disorder of lipid metabolism. Examples of such agentsinclude, but are not limited to an anti-inflammatory agent,anti-steatosis agent, anti-viral, and/or anti-fibrosis agent. Inaddition, other substances commonly used to protect the liver, such assilymarin, can also be used in conjunction with the iRNAs describedherein. Other agents useful for treating liver diseases includetelbivudine, entecavir, and protease inhibitors such as telaprevir andother disclosed, for example, in Tung et al., U.S. ApplicationPublication Nos. 2005/0148548, 2004/0167116, and 2003/0144217; and inHale et al., U.S. Application Publication No. 2004/0127488.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀.Compounds that exhibit high therapeutic indices are preferred.

The data obtained from cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofcompositions featured herein in the invention lies generally within arange of circulating concentrations that include the ED₅₀ with little orno toxicity. The dosage can vary within this range depending upon thedosage form employed and the route of administration utilized. For anycompound used in the methods featured in the invention, thetherapeutically effective dose can be estimated initially from cellculture assays. A dose can be formulated in animal models to achieve acirculating plasma concentration range of the compound or, whenappropriate, of the polypeptide product of a target sequence (e.g.,achieving a decreased concentration of the polypeptide) that includesthe IC₅₀ (i.e., the concentration of the test compound which achieves ahalf-maximal inhibition of symptoms) as determined in cell culture. Suchinformation can be used to more accurately determine useful doses inhumans. Levels in plasma can be measured, for example, by highperformance liquid chromatography.

In addition to their administration, as discussed above, the iRNAsfeatured in the invention can be administered in combination with otherknown agents effective in treatment of pathological processes mediatedby ANGPTL3 expression. In any event, the administering physician canadjust the amount and timing of iRNA administration on the basis ofresults observed using standard measures of efficacy known in the art ordescribed herein.

VI. Methods of the Invention

The present invention also provides methods of using an iRNA of theinvention and/or a composition containing an iRNA of the invention toreduce and/or inhibit ANGPTL3 expression in a cell. The methods includecontacting the cell with a dsRNA of the invention and maintaining thecell for a time sufficient to obtain degradation of the mRNA transcriptof an ANGPTL3gene, thereby inhibiting expression of the ANGPTL3 gene inthe cell. Reduction in gene expression can be assessed by any methodsknown in the art. For example, a reduction in the expression of ANGPTL3may be determined by determining the mRNA expression level of ANGPTL3using methods routine to one of ordinary skill in the art, e.g.,Northern blotting, qRT-PCR; by determining the protein level of ANGPTL3using methods routine to one of ordinary skill in the art, such asWestern blotting, immunological techniques. A reduction in theexpression of ANGPTL3 may also be assessed indirectly by measuring adecrease in biological activity of ANGPTL3, e.g., a decrease in thelevel of serum lipid, triglycerides, cholesterol and/or free fattyacids.

In the methods of the invention the cell may be contacted in vitro or invivo, i.e., the cell may be within a subject.

A cell suitable for treatment using the methods of the invention may beany cell that expresses an ANGPTL3gene. A cell suitable for use in themethods of the invention may be a mammalian cell, e.g., a primate cell(such as a human cell or a non-human primate cell, e.g., a monkey cellor a chimpanzee cell), a non-primate cell (such as a cow cell, a pigcell, a camel cell, a llama cell, a horse cell, a goat cell, a rabbitcell, a sheep cell, a hamster, a guinea pig cell, a cat cell, a dogcell, a rat cell, a mouse cell, a lion cell, a tiger cell, a bear cell,or a buffalo cell), a bird cell (e.g., a duck cell or a goose cell), ora whale cell. In one embodiment, the cell is a human cell, e.g., a humanliver cell.

ANGPTL3 expression is inhibited in the cell by at least about 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80,81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,99, or about 100%.

The in vivo methods of the invention may include administering to asubject a composition containing an iRNA, where the iRNA includes anucleotide sequence that is complementary to at least a part of an RNAtranscript of the ANGPTL3 gene of the mammal to be treated. When theorganism to be treated is a mammal such as a human, the composition canbe administered by any means known in the art including, but not limitedto oral, intraperitoneal, or parenteral routes, including intracranial(e.g., intraventricular, intraparenchymal and intrathecal), intravenous,intramuscular, subcutaneous, transdermal, airway (aerosol), nasal,rectal, and topical (including buccal and sublingual) administration.

In certain embodiments, the compositions are administered by intravenousinfusion or injection. In certain embodiments, the compositions areadministered by subcutaneous injection.

In some embodiments, the administration is via a depot injection. Adepot injection may release the iRNA in a consistent way over aprolonged time period. Thus, a depot injection may reduce the frequencyof dosing needed to obtain a desired effect, e.g., a desired inhibitionof ANGPTL3, or a therapeutic or prophylactic effect. A depot injectionmay also provide more consistent serum concentrations. Depot injectionsmay include subcutaneous injections or intramuscular injections. Inpreferred embodiments, the depot injection is a subcutaneous injection.

In some embodiments, the administration is via a pump. The pump may bean external pump or a surgically implanted pump. In certain embodiments,the pump is a subcutaneously implanted osmotic pump. In otherembodiments, the pump is an infusion pump. An infusion pump may be usedfor intravenous, subcutaneous, arterial, or epidural infusions. Inpreferred embodiments, the infusion pump is a subcutaneous infusionpump. In other embodiments, the pump is a surgically implanted pump thatdelivers the iRNA to the liver.

The mode of administration may be chosen based upon whether local orsystemic treatment is desired and based upon the area to be treated. Theroute and site of administration may be chosen to enhance targeting.

In one aspect, the present invention also provides methods forinhibiting the expression of an ANGPTL3 gene in a mammal. The methodsinclude administering to the mammal a composition comprising a dsRNAthat targets an ANGPTL3 gene in a cell of the mammal and maintaining themammal for a time sufficient to obtain degradation of the mRNAtranscript of the ANGPTL3 gene, thereby inhibiting expression of theANGPTL3 gene in the cell. Reduction in gene expression can be assessedby any methods known it the art and by methods, e.g. qRT-PCR, describedherein. Reduction in protein production can be assessed by any methodsknown it the art and by methods, e.g. ELISA, described herein. In oneembodiment, a puncture liver biopsy sample serves as the tissue materialfor monitoring the reduction in ANGPTL3 gene and/or protein expression.

The present invention further provides methods of treatment of a subjectin need thereof. The treatment methods of the invention includeadministering an iRNA of the invention to a subject, e.g., a subjectthat would benefit from a reduction and/or inhibition of ANGPTL3expression, in a therapeutically effective amount of an iRNA targetingan ANGPTL3 gene or a pharmaceutical composition comprising an iRNAtargeting an ANGPTL3 gene.

An iRNA of the invention may be administered as a “free iRNA.” A freeiRNA is administered in the absence of a pharmaceutical composition. Thenaked iRNA may be in a suitable buffer solution. The buffer solution maycomprise acetate, citrate, prolamine, carbonate, or phosphate, or anycombination thereof. In one embodiment, the buffer solution is phosphatebuffered saline (PBS). The pH and osmolarity of the buffer solutioncontaining the iRNA can be adjusted such that it is suitable foradministering to a subject.

Alternatively, an iRNA of the invention may be administered as apharmaceutical composition, such as a dsRNA liposomal formulation.

Subjects that would benefit from a reduction and/or inhibition ofANGPTL3 gene expression are those having a disorder of lipid metabolism,e.g., an inherited disorder of lipid metabolism or an acquired disorderof lipid metabolism. In one embodiment, a subject having disorder oflipid metabolism has hyperlipidemia. In another embodiment, a subjecthaving a disorder of lipid metabolism has hypertriglyceridemia.Treatment of a subject that would benefit from a reduction and/orinhibition of ANGPTL3 gene expression includes therapeutic treatment(e.g., a subject is having eruptive xanthomas) and prophylactictreatment (e.g., the subject is not having eruptive xanthomas or asubject may be at risk of developing eruptive xanthomas).

The invention further provides methods for the use of an iRNA or apharmaceutical composition thereof, e.g., for treating a subject thatwould benefit from reduction and/or inhibition of ANGPTL3 expression,e.g., a subject having a disorder of lipid metabolism, in combinationwith other pharmaceuticals and/or other therapeutic methods, e.g., withknown pharmaceuticals and/or known therapeutic methods, such as, forexample, those which are currently employed for treating thesedisorders. For example, in certain embodiments, an iRNA targetingANGPTL3 is administered in combination with, e.g., an agent useful intreating a disorder of lipid metabolism as described elsewhere herein.For example, additional agents suitable for treating a subject thatwould benefit from reducton in ANGPTL3 expression, e.g., a subjecthaving a disorder of lipid metabolism, may include agents that lower oneor more serum lipids. Non-limiting examples of such agents may includecholesterol synthesis inhibitors, such as HMG-CoA reductase inhibitors,e.g., statins. Statins may include atorvastatin (Lipitor), fluvastatin(Lescol), lovastatin (Mevacor), lovastatin extended-release (Altoprev),pitavastatin (Livalo), pravastatin (Pravachol), rosuvastatin (Crestor),and simvastatin (Zocor). Other agents useful in treating a disorder oflipid metabolism may include bile sequestering agents, such ascholestyramine and other resins; VLDL secretion inhibitors, such asniacin; lipophilic antioxidants, such as Probucol; acyl-CoA cholesterolacyl transferase inhibitors; farnesoid X receptor antagonists; sterolregulatory binding protein cleavage activating protein (SCAP)activators; microsomal triglyceride transfer protein (MTP) inhibitors;ApoE-related peptide; and therapeutic antibodies against ANGPTL3. Theadditional therapeutic agents may also include agents that raise highdensity lipoprotein (HDL), such as cholesteryl ester transfer protein(CETP) inhibitors. Furthermore, the additional therapeutic agents mayalso include dietary supplements, e.g., fish oil. The iRNA andadditional therapeutic agents may be administered at the same timeand/or in the same combination, e.g., parenterally, or the additionaltherapeutic agent can be administered as part of a separate compositionor at separate times and/or by another method known in the art ordescribed herein.

In one embodiment, the method includes administering a compositionfeatured herein such that expression of the target ANGPTL3 gene isdecreased, such as for about 1, 2, 3, 4, 5, 6, 7, 8, 12, 16, 18, 24hours, 28, 32, or about 36 hours. In one embodiment, expression of thetarget ANGPTL3 gene is decreased for an extended duration, e.g., atleast about two, three, four days or more, e.g., about one week, twoweeks, three weeks, or four weeks or longer.

Preferably, the iRNAs useful for the methods and compositions featuredherein specifically target RNAs (primary or processed) of the targetANGPTL3gene.

Compositions and methods for inhibiting the expression of these genesusing iRNAs can be prepared and performed as described herein.

Administration of the dsRNA according to the methods of the inventionmay result in a reduction of the severity, signs, symptoms, and/ormarkers of such diseases or disorders in a patient with a disorder oflipid metabolism. By “reduction” in this context is meant astatistically significant decrease in such level. The reduction can be,for example, at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or about 100%.

Efficacy of treatment or prevention of disease can be assessed, forexample by measuring disease progression, disease remission, symptomseverity, reduction in pain, quality of life, dose of a medicationrequired to sustain a treatment effect, level of a disease marker or anyother measurable parameter appropriate for a given disease being treatedor targeted for prevention. It is well within the ability of one skilledin the art to monitor efficacy of treatment or prevention by measuringany one of such parameters, or any combination of parameters. Forexample, efficacy of treatment of a disorder of lipid metabolism may beassessed, for example, by periodic monitoring of one or more serum lipidlevels. Comparisons of the later readings with the initial readingsprovide a physician an indication of whether the treatment is effective.It is well within the ability of one skilled in the art to monitorefficacy of treatment or prevention by measuring any one of suchparameters, or any combination of parameters. In connection with theadministration of an iRNA targeting ANGPTL3 or pharmaceuticalcomposition thereof, “effective against” a disorder of lipid metabolismindicates that administration in a clinically appropriate manner resultsin a beneficial effect for at least a statistically significant fractionof patients, such as a improvement of symptoms, a cure, a reduction indisease, extension of life, improvement in quality of life, or othereffect generally recognized as positive by medical doctors familiar withtreating disorder of lipid metabolisms and the related causes.

A treatment or preventive effect is evident when there is astatistically significant improvement in one or more parameters ofdisease status, or by a failure to worsen or to develop symptoms wherethey would otherwise be anticipated. As an example, a favorable changeof at least 10% in a measurable parameter of disease, and preferably atleast 20%, 30%, 40%, 50% or more can be indicative of effectivetreatment. Efficacy for a given iRNA drug or formulation of that drugcan also be judged using an experimental animal model for the givendisease as known in the art. When using an experimental animal model,efficacy of treatment is evidenced when a statistically significantreduction in a marker or symptom is observed.

Alternatively, the efficacy can be measured by a reduction in theseverity of disease as determined by one skilled in the art of diagnosisbased on a clinically accepted disease severity grading scale, as butone example the Child-Pugh score (sometimes the Child-Turcotte-Pughscore). Any positive change resulting in e.g., lessening of severity ofdisease measured using the appropriate scale, represents adequatetreatment using an iRNA or iRNA formulation as described herein.

Subjects can be administered a therapeutic amount of dsRNA, such asabout 0.01 mg/kg to about 5 mg/kg, about 0.01 mg/kg to about 10 mg/kg,about 0.05 mg/kg to about 5 mg/kg, about 0.05 mg/kg to about 10 mg/kg,about 0.1 mg/kg to about 5 mg/kg, about 0.1 mg/kg to about 10 mg/kg,about 0.2 mg/kg to about 5 mg/kg, about 0.2 mg/kg to about 10 mg/kg,about 0.3 mg/kg to about 5 mg/kg, about 0.3 mg/kg to about 10 mg/kg,about 0.4 mg/kg to about 5 mg/kg, about 0.4 mg/kg to about 10 mg/kg,about 0.5 mg/kg to about 5 mg/kg, about 0.5 mg/kg to about 10 mg/kg,about 1 mg/kg to about 5 mg/kg, about 1 mg/kg to about 10 mg/kg, about1.5 mg/kg to about 5 mg/kg, about 1.5 mg/kg to about 10 mg/kg, about 2mg/kg to about about 2.5 mg/kg, about 2 mg/kg to about 10 mg/kg, about 3mg/kg to about 5 mg/kg, about 3 mg/kg to about 10 mg/kg, about 3.5 mg/kgto about 5 mg/kg, about 4 mg/kg to about 5 mg/kg, about 4.5 mg/kg toabout 5 mg/kg, about 4 mg/kg to about 10 mg/kg, about 4.5 mg/kg to about10 mg/kg, about 5 mg/kg to about 10 mg/kg, about 5.5 mg/kg to about 10mg/kg, about 6 mg/kg to about 10 mg/kg, about 6.5 mg/kg to about 10mg/kg, about 7 mg/kg to about 10 mg/kg, about 7.5 mg/kg to about 10mg/kg, about 8 mg/kg to about 10 mg/kg, about 8.5 mg/kg to about 10mg/kg, about 9 mg/kg to about 10 mg/kg, or about 9.5 mg/kg to about 10mg/kg. Values and ranges intermediate to the recited values are alsointended to be part of this invention.

For example, the dsRNA may be administered at a dose of about 0.1, 0.2,0.3, 0.4, 0.5, 0.6, 0.7. 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,1.8. 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8. 2.9, 3, 3.1, 3.2,3.3, 3.4, 3.5, 3.6, 3.7, 3.8. 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7,4.8. 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8. 5.9, 6, 6.1, 6.2,6.3, 6.4, 6.5, 6.6, 6.7, 6.8. 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7,7.8. 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8. 8.9, 9, 9.1, 9.2,9.3, 9.4, 9.5, 9.6, 9.7, 9.8. 9.9, or about 10 mg/kg. Values and rangesintermediate to the recited values are also intended to be part of thisinvention.

In other embodiments, for example, when a composition of the inventioncomprises a dsRNA as described herein and an N-acetylgalactosamine,subjects can be administered a therapeutic amount of dsRNA, such as adose of about 0.1 to about 50 mg/kg, about 0.25 to about 50 mg/kg, about0.5 to about 50 mg/kg, about 0.75 to about 50 mg/kg, about 1 to about 50mg/mg, about 1.5 to about 50 mg/kb, about 2 to about 50 mg/kg, about 2.5to about 50 mg/kg, about 3 to about 50 mg/kg, about 3.5 to about 50mg/kg, about 4 to about 50 mg/kg, about 4.5 to about 50 mg/kg, about 5to about 50 mg/kg, about 7.5 to about 50 mg/kg, about 10 to about 50mg/kg, about 15 to about 50 mg/kg, about 20 to about 50 mg/kg, about 20to about 50 mg/kg, about 25 to about 50 mg/kg, about 25 to about 50mg/kg, about 30 to about 50 mg/kg, about 35 to about 50 mg/kg, about 40to about 50 mg/kg, about 45 to about 50 mg/kg, about 0.1 to about 45mg/kg, about 0.25 to about 45 mg/kg, about 0.5 to about 45 mg/kg, about0.75 to about 45 mg/kg, about 1 to about 45 mg/mg, about 1.5 to about 45mg/kb, about 2 to about 45 mg/kg, about 2.5 to about 45 mg/kg, about 3to about 45 mg/kg, about 3.5 to about 45 mg/kg, about 4 to about 45mg/kg, about 4.5 to about 45 mg/kg, about 5 to about 45 mg/kg, about 7.5to about 45 mg/kg, about 10 to about 45 mg/kg, about 15 to about 45mg/kg, about 20 to about 45 mg/kg, about 20 to about 45 mg/kg, about 25to about 45 mg/kg, about 25 to about 45 mg/kg, about 30 to about 45mg/kg, about 35 to about 45 mg/kg, about 40 to about 45 mg/kg, about 0.1to about 40 mg/kg, about 0.25 to about 40 mg/kg, about 0.5 to about 40mg/kg, about 0.75 to about 40 mg/kg, about 1 to about 40 mg/mg, about1.5 to about 40 mg/kb, about 2 to about 40 mg/kg, about 2.5 to about 40mg/kg, about 3 to about 40 mg/kg, about 3.5 to about 40 mg/kg, about 4to about 40 mg/kg, about 4.5 to about 40 mg/kg, about 5 to about 40mg/kg, about 7.5 to about 40 mg/kg, about 10 to about 40 mg/kg, about 15to about 40 mg/kg, about 20 to about 40 mg/kg, about 20 to about 40mg/kg, about 25 to about 40 mg/kg, about 25 to about 40 mg/kg, about 30to about 40 mg/kg, about 35 to about 40 mg/kg, about 0.1 to about 30mg/kg, about 0.25 to about 30 mg/kg, about 0.5 to about 30 mg/kg, about0.75 to about 30 mg/kg, about 1 to about 30 mg/mg, about 1.5 to about 30mg/kb, about 2 to about 30 mg/kg, about 2.5 to about 30 mg/kg, about 3to about 30 mg/kg, about 3.5 to about 30 mg/kg, about 4 to about 30mg/kg, about 4.5 to about 30 mg/kg, about 5 to about 30 mg/kg, about 7.5to about 30 mg/kg, about 10 to about 30 mg/kg, about 15 to about 30mg/kg, about 20 to about 30 mg/kg, about 20 to about 30 mg/kg, about 25to about 30 mg/kg, about 0.1 to about 20 mg/kg, about 0.25 to about 20mg/kg, about 0.5 to about 20 mg/kg, about 0.75 to about 20 mg/kg, about1 to about 20 mg/mg, about 1.5 to about 20 mg/kb, about 2 to about 20mg/kg, about 2.5 to about 20 mg/kg, about 3 to about 20 mg/kg, about 3.5to about 20 mg/kg, about 4 to about 20 mg/kg, about 4.5 to about 20mg/kg, about 5 to about 20 mg/kg, about 7.5 to about 20 mg/kg, about 10to about 20 mg/kg, or about 15 to about 20 mg/kg. Values and rangesintermediate to the recited values are also intended to be part of thisinvention.

For example, subjects can be administered a therapeutic amount of dsRNA,such as about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7. 0.8, 0.9, 1, 1.1, 1.2,1.3, 1.4, 1.5, 1.6, 1.7, 1.8. 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7,2.8. 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8. 3.9, 4, 4.1, 4.2,4.3, 4.4, 4.5, 4.6, 4.7, 4.8. 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7,5.8. 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8. 6.9, 7, 7.1, 7.2,7.3, 7.4, 7.5, 7.6, 7.7, 7.8. 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7,8.8. 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8. 9.9, 10.5, 11,11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18,18.5, 19, 19.5, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, orabout 50 mg/kg. Values and ranges intermediate to the recited values arealso intended to be part of this invention.

The iRNA can be administered by intravenous infusion over a period oftime, such as over a 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, or about a 25 minute period. The administrationmay be repeated, for example, on a regular basis, such as biweekly(i.e., every two weeks) for one month, two months, three months, fourmonths or longer. After an initial treatment regimen, the treatments canbe administered on a less frequent basis. For example, afteradministration biweekly for three months, administration can be repeatedonce per month, for six months or a year or longer. Administration ofthe iRNA can reduce ANGPTL3 levels, e.g., in a cell, tissue, blood,urine or other compartment of the patient by at least about 5%, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47, 48, 39, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80,81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,or at least about 99% or more.

Before administration of a full dose of the iRNA, patients can beadministered a smaller dose, such as a 5% infusion reaction, andmonitored for adverse effects, such as an allergic reaction. In anotherexample, the patient can be monitored for unwanted immunostimulatoryeffects, such as increased cytokine (e.g., TNF-alpha or INF-alpha)levels.

Alternatively, the iRNA can be administered subcutaneously, i.e., bysubcutaneous injection. One or more injections may be used to deliverthe desired daily dose of iRNA to a subject. The injections may berepeated over a period of time, such as over 2, 3, 4, 5, 6, 7, 8, 9, 10or 15 days. The administration may be repeated, for example, on aregular basis, such as biweekly (i.e., every two weeks) for one month,two months, three months, four months or longer. After an initialtreatment regimen, the treatments can be administered on a less frequentbasis. In some embodiments, a single dose of iRNA is followed by monthlydosing. In some embodiments, the dosing may comprise a loading phase ofmultiple doses on consecutive days.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the iRNAs and methods featured in the invention,suitable methods and materials are described below. All publications,patent applications, patents, and other references mentioned herein areincorporated by reference in their entirety. In case of conflict, thepresent specification, including definitions, will control. In addition,the materials, methods, and examples are illustrative only and notintended to be limiting.

EXAMPLES Example 1. iRNA Synthesis

Source of Reagents

Where the source of a reagent is not specifically given herein, suchreagent can be obtained from any supplier of reagents for molecularbiology at a quality/purity standard for application in molecularbiology.

Transcripts

siRNA design was carried out to identify siRNAs targeting the humanANGPTL3 transcript annotated in the NCBI Gene database(http://www.ncbi.nlm.nih.gov/gene/) and a cynomolgus monkey (Macacafascicularis; henceforth “cyno”) ANGPTL3 transcript produced viasequencing of cDNA prepared from liver RNA. Sequencing of cyno ANGPTL3mRNA was done in-house, and the mRNA sequence is shown in SEQ ID NO:9.Design used the following transcripts from the NCBI collection:Human—NM_014495.2 (SEQ ID NO:1); Mouse—NM_013913.3 (SEQ ID NO:2). AllsiRNA duplexes were designed that shared 100% identity with the listedhuman and cyno transcripts. A subset of siRNA duplexes, described below,also shared 100% identity with the mouse (Mus musculus) ANGPTL3transcript found in NCBI Gene database.

siRNA Design, Specificity, and Efficacy Prediction

The predicted specificity of all possible 19mers was predicted from eachsequence. Candidate 19mers were then selected that lacked repeats longerthan 7 nucleotides. These 977 candidate human/cyno siRNAs, and a subsetof 38 that also matched mouse (“human/cyno/mouse candidate siRNAs”) werethen used in a comprehensive search against the human transcriptome(defined as the set of NM_ and XM_records within the human NCBI Refseqset) using an exhaustive “brute-force” algorithm implemented in thepython script ‘BruteForce.py’. The script next parsed thetranscript-oligo alignments to generate a score based on the positionand number of mismatches between the siRNA and any potential‘off-target’ transcript. The off-target score is weighted to emphasizedifferences in the ‘seed’ region of siRNAs, in positions 2-9 from the 5′end of the molecule. Each oligo-transcript pair from the brute-forcesearch was given a mismatch score by summing the individual mismatchscores; mismatches in the position 2-9 were counted as 2.8, mismatchesin the cleavage site positions 10-11 were counted as 1.2, and mismatchesin region 12-19 counted as 1.0. An additional off-target prediction wascarried out by comparing the frequency of heptamers and octomers derivedfrom 3 distinct, seed-derived hexamers of each oligo. The hexamers frompositions 2-7 relative to the 5′ start were used to create 2 heptamersand one octomer. ‘Heptamer1’ was created by adding a 3′ A to thehexamer; ‘heptamer2’ was created by adding a 5′ A to the hexamer;octomer was created by adding an A to both 5′ and 3′ ends of thehexamer. The frequency of octomers and heptamers in the human 3′UTRome(defined as the subsequence of the transcriptome from NCBI's Refseqdatabase where the end of the coding region, the ‘CDS’, is clearlydefined) was pre-calculated. The octomer frequency was normalized to theheptamer frequency using the median value from the range of octomerfrequencies. A ‘mirSeedScore’ was then calculated by calculating the sumof ((3× normalized octomer count)+(2× heptamer2 count)+(1× heptamer1count)).

Both siRNAs strands were assigned to a category of specificity accordingto the calculated scores: a score above 3 qualifies as highly specific,equal to 3 as specific and between 2.2 and 2.8 as moderately specific.Sorting was carried out by the specificity of the antisense strand.Duplexes were then selected from the human/cyno set with antisenseoligos lacking miRNA seed matches, scores of 3 or better, less than 65%overall GC content, no GC at the first position, 4 or more Us or As inthe seed region, and GC at the nineteenth position. Duplexes from thehuman/cyno/mouse set with antisense oligos having scores of 2 or better,less than 65% overall GC content, and no GC at the first position werealso selected.

siRNA Sequence Selection

A total of 47 sense and 47 antisense derived siRNA oligos from thehuman/cyno set were synthesized and formed into duplexes. A total of 15sense and 15 antisense derived siRNAs from the human/cyno/mouse set weresynthesized and formed into duplexes.

Synthesis of ANGPTL3 Sequences

ANGPTL3 sequences were synthesized on a MerMade 192 synthesizer ateither a 1 or 0.2 μmol scale. Single strands were synthesized with2′O-methyl modifications for transfection based in vitro screening. Foruse in free uptake screening assays, 3′ GalNAc conjugates were made with2′F and 2′-O-methyl chemical modifications. In these designs, GalNAcmoiety was placed at the 3′end of the sense strand. The antisensesequence was 23 nucleotides in length and also contained 2′F and2′Omethyl chemical modifications with two phosphorothioate linkages atthe 3′end.

On one set of 21mer single strands and duplexes, ‘endolight’ chemistrywas applied as detailed below.

-   -   All pyrimidines (cytosine and uridine) in the sense strand were        modified with 2′-O-Methyl nucleotides (2′ O-Methyl C and        2′-O-Methyl U)    -   In the antisense strand, pyrimidines adjacent (towards 5′        position) to ribo A nucleoside were replaced with their        corresponding 2′-O-Methyl nucleosides    -   A two base dTsdT extension at the 3′ end of both sense and anti        sense sequences was introduced

For GalNAc conjugated 21mer sense and complementary 23mer antisensesequences, 2′F and 2′OMethyl modified single strands were synthesized.The synthesis was performed on a GalNAc modified CPG support for thesense strand and CPG modified with universal support for the antisensesequence at a 1 μmol scale. The sequence motif named TOFFEE was applied,in which the sense strand contained a three-nucleotide 2′F-modifiedmotif at positions 9, 10 and 11 and in the antisense, a2′OMethyl-modified motif was included at positions 11, 12 and 13.

Synthesis, Cleavage and Deprotection

The synthesis of ANGPTL3 sequences used solid supported oligonucleotidesynthesis using phosphoramidite chemistry. For 21 mer endolightsequences, a deoxy thymidine CPG was used as the solid support while forthe GalNAc conjugates, GalNAc solid support for the sense strand and auniversal CPG for the antisesense strand were used.

The synthesis of the above sequences was performed at either a 1 or 0.2μm scale in 96 well plates. The amidite solutions were prepared at 0.1Mconcentration and ethyl thio tetrazole (0.6M in Acetonitrile) was usedas the activator.

The synthesized sequences were cleaved and deprotected in 96 wellplates, using methylamine in the first step and fluoride reagent in thesecond step. For GalNAc and 2′F nucleoside containing sequences,deprotection conditions were modified. Sequences after cleavage anddeprotection were precipitated using an acetone:ethanol (80:20) mix andthe pellets were re-suspended in 0.2M sodium acetate buffer. Samplesfrom each sequence were analyzed by LC-MS to confirm the identity, UVfor quantification and a selected set of samples by IEX chromatographyto determine purity.

Purification, Desalting and Annealing

ANGPTL3 sequences were precipitated and purified on an AKTA Purifiersystem using a Sephadex column. The ANGPTL3 was run at ambienttemperature. Sample injection and collection was performed in 96 wellplates with 1.8 mL deep wells. A single peak corresponding to the fulllength sequence was collected in the eluent. The desalted ANGPTL3sequences were analyzed for concentration (by UV measurement at A₂₆₀)and purity (by ion exchange HPLC). The complementary single strands werethen combined in a 1:1 stoichiometric ratio to form siRNA duplexes.

Example 2. In Vitro Screening

Cell Culture and Transfections

Hep3B cells (ATCC, Manassas, Va.) were grown to near confluence at 37°C. in an atmosphere of 5% CO₂ in RPMI (ATCC) supplemented with 10% FBS,streptomycin, and glutamine (ATCC) before being released from the plateby trypsinization. Transfection was carried out by adding 14.8 μl ofOpti-MEM plus 0.2 μl of Lipofectamine RNAiMax per well (Invitrogen,Carlsbad Calif. cat #13778-150) to 5 μl of siRNA duplexes per well intoa 96-well plate and incubated at room temperature for 15 minutes. 80 μlof complete growth media without antibiotic containing ˜2×10⁴ Hep3Bcells were then added to the siRNA mixture. Cells were incubated foreither 24 or 120 hours prior to RNA purification. Single doseexperiments were performed at 10 nM and 0.1 nM final duplexconcentration and dose response experiments were done at 10, 1, 0.5,0.1, 0.05, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005 and 0.00001 nMfinal duplex concentration unless otherwise stated.

Free Uptake Transfection

5 μl of each GalNac conjugated siRNA in PBS was combined with 4×10⁴freshly thawed cryopreserved Cynomolgus monkey hepatocytes resuspendedin 95 μl of In Vitro Gro CP media (In Vitro Technologies—Celsis,Baltimore, Md.) in each well of a 96 well plate. The mixture wasincubated for about 24 hrs at 37° C. in an atmosphere of 5% CO₂. siRNAswere tested at final concentrations of 500 nM, 100 nM and 10 nM forefficacy free uptake assays. For dose response screens, final siRNAconcentrations were 500 nM, 100 nM, 20 nM, 4 nM, 0.8 nM, 0.16 nM, 0.032nM and 0.0064 nM.

Total RNA Isolation Using DYNABEADS mRNA Isolation Kit (Invitrogen, Part#: 610-12)

Cells were harvested and lysed in 150 μl of Lysis/Binding Buffer thenmixed for 5 minute at 850 rpm using an Eppendorf Thermomixer (the mixingspeed was the same throughout the process). Ten microliters of magneticbeads and 80 μl of Lysis/Binding Buffer mixture were added to a roundbottom plate and mixed for 1 minute. Magnetic beads were captured usingmagnetic stand and the supernatant was removed without disturbing thebeads. After removing supernatant, the lysed cells were added to theremaining beads and mixed for 5 minutes. After removing supernatant,magnetic beads were washed 2 times with 150 μl Wash Buffer A and mixedfor 1 minute. Beads were captured again and supernatant removed. Beadswere then washed with 150 μl of Wash Buffer B, captured, and thesupernatant was removed. Beads were next washed with 150 μl ElutionBuffer, captured, and the supernatant was removed. Beads were allowed todry for 2 minutes. After drying, 50 μl of Elution Buffer was added andmixed for 5 minutes at 70° C. Beads were captured on magnet for 5minutes. 40 μl of supernatant was removed and added to another 96 wellplate.

cDNA Synthesis Using ABI High Capacity cDNA Reverse Transcription Kit(Applied Biosystems, Foster City, Calif., Cat #4368813)

A master mix of 2 μl 10× Buffer, 0.8 μl 25× dNTPs, 2 μl Random primers,1 μl Reverse Transcriptase, 1 μl RNase inhibitor and 3.2 μl of H₂O perreaction were added into 10 μl total RNA. cDNA was generated using aBio-Rad C-1000 or S-1000 thermal cycler (Hercules, Calif.) through thefollowing steps: 25° C. 10 min, 37° C. 120 min, 85° C. 5 sec, 4° C.hold.

Real Time PCR

2 μl of cDNA was added to a master mix containing 0.5 μl GAPDH TaqManProbe (Applied Biosystems Cat #4326317E), 0.5 μl ANGPTL TaqMan probe(Applied Biosystems cat #Hs00205581_ml) and 5 μl Lightcycler 480 probemaster mix (Roche Cat #04887301001) per well in a 384 well 50 plates(Roche cat #04887301001). Real time PCR was done in an ABI 7900HT RealTime PCR system (Applied Biosystems) using the ΔΔCt(RQ) assay. Eachduplex was tested in two independent transfections, and eachtransfection was assayed in duplicate, unless otherwise noted in thesummary tables.

To calculate relative fold change, real time data was analyzed using theΔΔCt method and normalized to assays performed with cells transfectedwith 10 nM AD-1955, or mock transfected cells. IC₅₀s were calculatedusing a 4 parameter fit model using XLFit and normalized to cellstransfected with AD-1955 or naïve cells over the same dose range, or toits own lowest dose. AD-1955 sequence, used as a negative control,targets luciferase and has the following sequence: sense:cuuAcGcuGAGuAcuucGAdTsdT (SEQ ID NO: 14); antisense:UCGAAGuACUcAGCGuAAGdTsdT (SEQ ID NO: 15).

Viability Screens

Cell viability was measured on days 3 and 6 in HeLa and Hep3B cellsfollowing transfection with 10, 1, 0.5, 0.1, 0.05 nM siRNA. Cells wereplated at a density of 10,000 cells per well in 96 well plates. EachsiRNA was assayed in triplicate and the data averaged. siRNAs targetingPLK1 and AD-19200 were included as positive controls for loss ofviability, and AD-1955 and mock transfected cells as negative controls.PLK1 and AD-19200 result in a dose dependent loss of viability. Tomeasure viability, 20 μl of CellTiter Blue (Promega) was added to eachwell of the 96 well plates after 3 or 6 days and incubated at 37° C. for2 hours. Plates were then read in a Spectrophotometer (MolecularDevices) at 560Ex/590Em. Viability was expressed as the average value oflight units from three replicate transfections30 /−standard deviation.Relative viability was assessed by first averaging the three replicatetransfections and then normalizing Mock transfected cells. Data isexpressed as % viabile cells.

TABLE 1 Abbreviations of nucleotide monomers used in nucleic acidsequence representation. It will be understood that these monomers, whenpresent in an oligonucleotide, are mutually linked by5′-3′-phosphodiester bonds. Abbreviation Nucleotide(s) A adenosine Ccytidine G guanosine T thymidine U uridine N any nucleotide (G, A, C, Tor U) a 2′-O-methyladenosine c 2′-O-methylcytidine g2′-O-methylguanosine u 2′-O-methyluridine dT 2′-deoxythymidine sphosphorothioate linkage

TABLE 2Unmodified sense and antisense strand sequences of ANGPTL3 dsRNAsSense Sequence Antisense Sequence (SEQ ID NOS 16-77, (SEQ ID NOS 78-139,Duplex Sense respectively, in  Position in Antisense respectively, inPosition in ID Name order of appearance) NM_014495.2 Nameorder of appearance) NM _014495.2 AD-45939.1 A-96225.1UAUUUGAUCAGUCUUUUUA  281-299 A-96226.1 UAAAAAGACUGAUCAAAUA  281-299AD-45858.1 A-96149.1 GAGCAACUAACUAACUUAA  478-496 A-96150.1UUAAGUUAGUUAGUUGCUC  478-496 AD-45869.1 A-96137.1 GGCCAAAUUAAUGACAUAU 247-265 A-96138.1 AUAUGUCAUUAAUUUGGCC  247-265 AD-45884.1 A-96189.1CGAAUUGAGUUGGAAGACU 1045-1063 A-96190.1 AGUCUUCCAACUCAAUUCG 1045-1063AD-45892.1 A-96129.1 CCUCCUUCAGUUGGGACAU  198-216 A-96130.1AUGUCCCAACUGAAGGAGG  198-216 AD-45899.1 A-96147.1 CACUUGAACUCAACUCAAA 401-419 A-96148.1 UUUGAGUUGAGUUCAAGUG  401-419 AD-45915.1 A-96231.1GUCCAUGGACAUUAAUUCA  890-908 A-96232.1 UGAAUUAAUGUCCAUGGAC  890-908AD-45924.1 A-96219.1 AAUCAAGAUUUGCUAUGUU  152-170 A-96220.1AACAUAGCAAAUCUUGAUU  152-170 AD-45860.1 A-96181.1 CUAGAGAAGAUAUACUCCA1000-1018 A-96182.1 UGGAGUAUAUCUUCUCUAG 1000-1018 AD-45870.1 A-96153.1CUAACUAACUUAAUUCAAA  484-502 A-96154.1 UUUGAAUUAAGUUAGUUAG  484-502AD-45870.2 A-96153.2 CUAACUAACUUAAUUCAAA  484-502 A-96154.2UUUGAAUUAAGUUAGUUAG  484-502 AD-45877.1 A-96171.1 CAUUAAUUCAACAUCGAAU 899-917 A-96172.1 AUUCGAUGUUGAAUUAAUG  899-917 AD-45885.1 A-96205.1CAAAAUGUUGAUCCAUCCA 1392-1410 A-96206.1 UGGAUGGAUCAACAUUUUG 1392-1410AD-45893.1 A-96145.1 CAUAUAAACUACAAGUCAA  359-377 A-96146.1UUGACUUGUAGUUUAUAUG  359-377 AD-45900.1 A-96163.1 GACCCAGCAACUCUCAAGU 839-857 A-96164.1 ACUUGAGAGUUGCUGGGUC  839-857 AD-45925.1 A-96235.1GGUUGGGCCUAGAGAAGAU  992-1010 A-96236.1 AUCUUCUCUAGGCCCAACC  992-1010AD-45861.1 A-96197.1 GUGUGGAGAAAACAACCUA 1272-1290 A-96198.1UAGGUUGUUUUCUCCACAC 1272-1290 AD-45871.1 A-96169.1 GACAUUAAUUCAACAUCGA 897-915 A-96170.1 UCGAUGUUGAAUUAAUGUC  897-915 AD-45878.1 A-96187.1CAUAGUGAAGCAAUCUAAU 1017-1035 A-96188.1 AUUAGAUUGCUUCACUAUG 1017-1035AD-45886.1 A-96127.1 CUAUGUUAGACGAUGUAAA  164-182 A-96128.1UUUACAUCGUCUAACAUAG  164-182 AD-45894.1 A-96161.1 CACAGAAAUUUCUCUAUCU 684-702 A-96162.1 AGAUAGAGAAAUUUCUGUG  684-702 AD-45901.1 A-96179.1GUUGGGCCUAGAGAAGAUA  993-1011 A-96180.1 UAUCUUCUCUAGGCCCAAC  993-1011AD-45909.1 A-96213.1 GCCAAAAUCAAGAUUUGCU  147-165 A-96214.1AGCAAAUCUUGAUUUUGGC  147-165 AD-45934.1 A-96223.1 ACAUAUUUGAUCAGUCUUU 278-296 A-96224.1 AAAGACUGAUCAAAUAUGU  278-296 AD-45934.2 A-96223.2ACAUAUUUGAUCAGUCUUU  278-296 A-96224.2 AAAGACUGAUCAAAUAUGU  278-296AD-45863.1 A-96135.1 CUUAAAGACUUUGUCCAUA  220-238 A-96136.1UAUGGACAAAGUCUUUAAG  220-238 AD-45872.1 A-96185.1 CCAUAGUGAAGCAAUCUAA1016-1034 A-96186.1 UUAGAUUGCUUCACUAUGG 1016-1034 AD-45879.1 A-96203.1CAACCAAAAUGUUGAUCCA 1388-1406 A-96204.1 UGGAUCAACAUUUUGGUUG 1388-1406AD-45887.1 A-96143.1 CUACAUAUAAACUACAAGU  356-374 A-96144.1ACUUGUAGUUUAUAUGUAG  356-374 AD-45895.1 A-96177.1 GGGAGGCUUGAUGGAGAAU 970-988 A-96178.1 AUUCUCCAUCAAGCCUCCC  970-988 AD-45902.1 A-96195.1GGUGUUUUCUACUUGGGAU 1188-1206 A-96196.1 AUCCCAAGUAGAAAACACC 1188-1206AD-45910.1 A-96229.1 AAGAGCACCAAGAACUACU  711-729 A-96230.1AGUAGUUCUUGGUGCUCUU  711-729 AD-45935.1 A-96239.1 UGGAGAAAACAACCUAAAU1275-1293 A-96240.1 AUUUAGGUUGUUUUCUCCA 1275-1293 AD-45864.1 A-96151.1GCAACUAACUAACUUAAUU  480-498 A-96152.1 AAUUAAGUUAGUUAGUUGC  480-498AD-45873.1 A-96201.1 CAACCUAAAUGGUAAAUAU 1284-1302 A-96202.1AUAUUUACCAUUUAGGUUG 1284-1302 AD-45880.1 A-96125.1 GCUAUGUUAGACGAUGUAA 163-181 A-96126.1 UUACAUCGUCUAACAUAGC  163-181 AD-45888.1 A-96159.1CCCACAGAAAUUUCUCUAU  682-700 A-96160.1 AUAGAGAAAUUUCUGUGGG  682-700AD-45896.1 A-96193.1 GAUUUGGUGUUUUCUACUU 1183-1201 A-96194.1AAGUAGAAAACACCAAAUC 1183-1201 AD-45903.1 A-96211.1 CAGAGCCAAAAUCAAGAUU 143-161 A-96212.1 AAUCUUGAUUUUGGCUCUG  143-161 AD-45919.1 A-96217.1AAAUCAAGAUUUGCUAUGU  151-169 A-96218.1 ACAUAGCAAAUCUUGAUUU  151-169AD-45865.1 A-96167.1 CAUGGACAUUAAUUCAACA  893-911 A-96168.1UGUUGAAUUAAUGUCCAUG  893-911 AD-45874.1 A-96123.1 GAUUUGCUAUGUUAGACGA 158-176 A-96124.1 UCGUCUAACAUAGCAAAUC  158-176 AD-45881.1 A-96141.1GAACUACAUAUAAACUACA  353-371 A-96142.1 UGUAGUUUAUAUGUAGUUC  353-371AD-45889.1 A-96175.1 CGAAUAGAUGGAUCACAAA  913-931 A-96176.1UUUGUGAUCCAUCUAUUCG  913-931 AD-45897.1 A-96209.1 CUUGUUAAAACUCUAAACU1817-1835 A-96210.1 AGUUUAGAGUUUUAACAAG 1817-1835 AD-45904.1 A-96227.1AUUUGAUCAGUCUUUUUAU  282-300 A-96228.1 AUAAAAAGACUGAUCAAAU  282-300AD-45920.1 A-96233.1 UCCAUGGACAUUAAUUCAA  891-909 A-96234.1UUGAAUUAAUGUCCAUGGA  891-909 AD-45856.1 A-96117.1 CACAAUUAAGCUCCUUCUU  57-75 A-96118.1 AAGAAGGAGCUUAAUUGUG   57-75 AD-45929.1 A-96221.1CAACAUAUUUGAUCAGUCU  276-294 A-96222.1 AGACUGAUCAAAUAUGUUG  276-294AD-45866.1 A-96183.1 CUCCAUAGUGAAGCAAUCU 1014-1032 A-96184.1AGAUUGCUUCACUAUGGAG 1014-1032 AD-45875.1 A-96139.1 GCCAAAUUAAUGACAUAUU 248-266 A-96140.1 AAUAUGUCAUUAAUUUGGC  248-266 AD-45882.1 A-96157.1CAACAGCAUAGUCAAAUAA  622-640 A-96158.1 UUAUUUGACUAUGCUGUUG  622-640AD-45890.1 A-96191.1 GGAAAUCACGAAACCAACU 1105-1123 A-96192.1AGUUGGUUUCGUGAUUUCC 1105-1123 AD-45898.1 A-96131.1 CAGUUGGGACAUGGUCUUA 205-223 A-96132.1 UAAGACCAUGUCCCAACUG  205-223 AD-45857.1 A-96133.1GACAUGGUCUUAAAGACUU  212-230 A-96134.1 AAGUCUUUAAGACCAUGUC  212-230AD-45930.1 A-96237.1 UGUGGAGAAAACAACCUAA 1273-1291 A-96238.1UUAGGUUGUUUUCUCCACA 1273-1291 AD-45867.1 A-96199.1 GUGGAGAAAACAACCUAAA1274-1292 A-96200.1 UUUAGGUUGUUUUCUCCAC 1274-1292 AD-45876.1 A-96155.1CCAACAGCAUAGUCAAAUA  621-639 A-96156.1 UAUUUGACUAUGCUGUUGG  621-639AD-45883.1 A-96173.1 CAACAUCGAAUAGAUGGAU  907-925 A-96174.1AUCCAUCUAUUCGAUGUUG  907-925 AD-45891.1 A-96207.1 GCAAAUUUAAAAGGCAAUA1441-1459 A-96208.1 UAUUGCCUUUUAAAUUUGC 1441-1459 AD-45914.1 A-96215.1CAAAAUCAAGAUUUGCUAU  149-167 A-96216.1 AUAGCAAAUCUUGAUUUUG  149-167AD-15838.1 A-26242.1 AGAGCCAAAAUCAAGAUUU  144-162 A-26243.2AAAUCUUGAUUUUGGCUCU  144-162

TABLE 3 Modified sense and antisense strand sequences of ANGPTL3 dsRNAsSense Sequence Antisense Sequence (SEQ ID NOS 140-201,(SEQ ID NOS 202-263, Duplex Sense respectively, in Antisense respectively, in ID OligoName order of appearance) OligoNameorder of appearance) AD-45939.1 A-96225.1 uAuuuGAucAGucuuuuuAdTsdTA-96226.1 uAAAAAGACUGAUcAAAuAdTsdT AD-45858.1 A-96149.1GAGcAAcuAAcuAAcuuAAdTsdT A-96150.1 UuAAGUuAGUuAGUUGCUCdTsdT AD-45869.1A-96137.1 GGccAAAuuAAuGAcAuAudTsdT A-96138.1 AuAUGUcAUuAAUUUGGCCdTsdTAD-45884.1 A-96189.1 cGAAuuGAGuuGGAAGAcudTsdT A-96190.1AGUCUUCcAACUcAAUUCGdTsdT AD-45892.1 A-96129.1 ccuccuucAGuuGGGAcAudTsdTA-96130.1 AUGUCCcAACUGAAGGAGGdTsdT AD-45899.1 A-96147.1cAcuuGAAcucAAcucAAAdTsdT A-96148.1 UUUGAGUUGAGUUcAAGUGdTsdT AD-45915.1A-96231.1 GuccAuGGAcAuuAAuucAdTsdT A-96232.1 UGAAUuAAUGUCcAUGGACdTsdTAD-45924.1 A-96219.1 AAucAAGAuuuGcuAuGuudTsdT A-96220.1AAcAuAGcAAAUCUUGAUUdTsdT AD-45860.1 A-96181.1 cuAGAGAAGAuAuAcuccAdTsdTA-96182.1 UGGAGuAuAUCUUCUCuAGdTsdT AD-45870.1 A-96153.1cuAAcuAAcuuAAuucAAAdTsdT A-96154.1 UUUGAAUuAAGUuAGUuAGdTsdT AD-45870.2A-96153.2 cuAAcuAAcuuAAuucAAAdTsdT A-96154.2 UUUGAAUuAAGUuAGUuAGdTsdTAD-45877.1 A-96171.1 cAuuAAuucAAcAucGAAudTsdT A-96172.1AUUCGAUGUUGAAUuAAUGdTsdT AD-45885.1 A-96205.1 cAAAAuGuuGAuccAuccAdTsdTA-96206.1 UGGAUGGAUcAAcAUUUUGdTsdT AD-45893.1 A-96145.1cAuAuAAAcuAcAAGucAAdTsdT A-96146.1 UUGACUUGuAGUUuAuAUGdTsdT AD-45900.1A-96163.1 GAcccAGcAAcucucAAGudTsdT A-96164.1 ACUUGAGAGUUGCUGGGUCdTsdTAD-45925.1 A-96235.1 GGuuGGGccuAGAGAAGAudTsdT A-96236.1AUCUUCUCuAGGCCcAACCdTsdT AD-45861.1 A-96197.1 GuGuGGAGAAAAcAAccuAdTsdTA-96198.1 uAGGUUGUUUUCUCcAcACdTsdT AD-45871.1 A-96169.1GAcAuuAAuucAAcAucGAdTsdT A-96170.1 UCGAUGUUGAAUuAAUGUCdTsdT AD-45878.1A-96187.1 cAuAGuGAAGcAAucuAAudTsdT A-96188.1 AUuAGAUUGCUUcACuAUGdTsdTAD-45886.1 A-96127.1 cuAuGuuAGAcGAuGuAAAdTsdT A-96128.1UUuAcAUCGUCuAAcAuAGdTsdT AD-45894.1 A-96161.1 cAcAGAAAuuucucuAucudTsdTA-96162.1 AGAuAGAGAAAUUUCUGUGdTsdT AD-45901.1 A-96179.1GuuGGGccuAGAGAAGAuAdTsdT A-96180.1 uAUCUUCUCuAGGCCcAACdTsdT AD-45909.1A-96213.1 GccAAAAucAAGAuuuGcudTsdT A-96214.1 AGcAAAUCUUGAUUUUGGCdTsdTAD-45934.1 A-96223.1 AcAuAuuuGAucAGucuuudTsdT A-96224.1AAAGACUGAUcAAAuAUGUdTsdT AD-45934.2 A-96223.2 AcAuAuuuGAucAGucuuudTsdTA-96224.2 AAAGACUGAUcAAAuAUGUdTsdT AD-45863.1 A-96135.1cuuAAAGAcuuuGuccAuAdTsdT A-96136.1 uAUGGAcAAAGUCUUuAAGdTsdT AD-45872.1A-96185.1 ccAuAGuGAAGcAAucuAAdTsdT A-96186.1 UuAGAUUGCUUcACuAUGGdTsdTAD-45879.1 A-96203.1 cAAccAAAAuGuuGAuccAdTsdT A-96204.1UGGAUcAAcAUUUUGGUUGdTsdT AD-45887.1 A-96143.1 cuAcAuAuAAAcuAcAAGudTsdTA-96144.1 ACUUGuAGUUuAuAUGuAGdTsdT AD-45895.1 A-96177.1GGGAGGcuuGAuGGAGAAudTsdT A-96178.1 AUUCUCcAUcAAGCCUCCCdTsdT AD-45902.1A-96195.1 GGuGuuuucuAcuuGGGAudTsdT A-96196.1 AUCCcAAGuAGAAAAcACCdTsdTAD-45910.1 A-96229.1 AAGAGcAccAAGAAcuAcudTsdT A-96230.1AGuAGUUCUUGGUGCUCUUdTsdT AD-45935.1 A-96239.1 uGGAGAAAAcAAccuAAAudTsdTA-96240.1 AUUuAGGUUGUUUUCUCcAdTsdT AD-45864.1 A-96151.1GcAAcuAAcuAAcuuAAuudTsdT A-96152.1 AAUuAAGUuAGUuAGUUGCdTsdT AD-45873.1A-96201.1 cAAccuAAAuGGuAAAuAudTsdT A-96202.1 AuAUUuACcAUUuAGGUUGdTsdTAD-45880.1 A-96125.1 GcuAuGuuAGAcGAuGuAAdTsdT A-96126.1UuAcAUCGUCuAAcAuAGCdTsdT AD-45888.1 A-96159.1 cccAcAGAAAuuucucuAudTsdTA-96160.1 AuAGAGAAAUUUCUGUGGGdTsdT AD-45896.1 A-96193.1GAuuuGGuGuuuucuAcuudTsdT A-96194.1 AAGuAGAAAAcACcAAAUCdTsdT AD-45903.1A-96211.1 cAGAGccAAAAucAAGAuudTsdT A-96212.1 AAUCUUGAUUUUGGCUCUGdTsdTAD-45919.1 A-96217.1 AAAucAAGAuuuGcuAuGudTsdT A-96218.1AcAuAGcAAAUCUUGAUUUdTsdT AD-45865.1 A-96167.1 cAuGGAcAuuAAuucAAcAdTsdTA-96168.1 UGUUGAAUuAAUGUCcAUGdTsdT AD-45874.1 A-96123.1GAuuuGcuAuGuuAGAcGAdTsdT A-96124.1 UCGUCuAAcAuAGcAAAUCdTsdT AD-45881.1A-96141.1 GAAcuAcAuAuAAAcuAcAdTsdT A-96142.1 UGuAGUUuAuAUGuAGUUCdTsdTAD-45889.1 A-96175.1 cGAAuAGAuGGAucAcAAAdTsdT A-96176.1UUUGUGAUCcAUCuAUUCGdTsdT AD-45897.1 A-96209.1 cuuGuuAAAAcucuAAAcudTsdTA-96210.1 AGUUuAGAGUUUuAAcAAGdTsdT AD-45904.1 A-96227.1AuuuGAucAGucuuuuuAudTsdT A-96228.1 AuAAAAAGACUGAUcAAAUdTsdT AD-45920.1A-96233.1 uccAuGGAcAuuAAuucAAdTsdT A-96234.1 UUGAAUuAAUGUCcAUGGAdTsdTAD-45856.1 A-96117.1 cAcAAuuAAGcuccuucuudTsdT A-96118.1AAGAAGGAGCUuAAUUGUGdTsdT AD-45929.1 A-96221.1 cAAcAuAuuuGAucAGucudTsdTA-96222.1 AGACUGAUcAAAuAUGUUGdTsdT AD-45866.1 A-96183.1cuccAuAGuGAAGcAAucudTsdT A-96184.1 AGAUUGCUUcACuAUGGAGdTsdT AD-45875.1A-96139.1 GccAAAuuAAuGAcAuAuudTsdT A-96140.1 AAuAUGUcAUuAAUUUGGCdTsdTAD-45882.1 A-96157.1 cAAcAGcAuAGucAAAuAAdTsdT A-96158.1UuAUUUGACuAUGCUGUUGdTsdT AD-45890.1 A-96191.1 GGAAAucAcGAAAccAAcudTsdTA-96192.1 AGUUGGUUUCGUGAUUUCCdTsdT AD-45898.1 A-96131.1cAGuuGGGAcAuGGucuuAdTsdT A-96132.1 uAAGACcAUGUCCcAACUGdTsdT AD-45857.1A-96133.1 GAcAuGGucuuAAAGAcuudTsdT A-96134.1 AAGUCUUuAAGACcAUGUCdTsdTAD-45930.1 A-96237.1 uGuGGAGAAAAcAAccuAAdTsdT A-96238.1UuAGGUUGUUUUCUCcAcAdTsdT AD-45867.1 A-96199.1 GuGGAGAAAAcAAccuAAAdTsdTA-96200.1 UUuAGGUUGUUUUCUCcACdTsdT AD-45876.1 A-96155.1ccAAcAGcAuAGucAAAuAdTsdT A-96156.1 uAUUUGACuAUGCUGUUGGdTsdT AD-45883.1A-96173.1 cAAcAucGAAuAGAuGGAudTsdT A-96174.1 AUCcAUCuAUUCGAUGUUGdTsdTAD-45891.1 A-96207.1 GcAAAuuuAAAAGGcAAuAdTsdT A-96208.1uAUUGCCUUUuAAAUUUGCdTsdT AD-45914.1 A-96215.1 cAAAAucAAGAuuuGcuAudTsdTA-96216.1 AuAGcAAAUCUUGAUUUUGdTsdT AD-15838.1 A-26242.1AGAGccAAAAucAAGAuuudTsdT A-26243.2 AAAUCUuGAUUUuGGCUCUdTsdT Lowercasenucleotides (a, u, g, c) are 2'-0-methyl nucleotides; s is aphosphothiorate linkage.

TABLE 4 Results of single dose screen using ANGPTL3 dsRNA sequences Theexperiments were conducted using modified oligonucleotide duplexeslisted in Table 3. The sequence of AD-15838.2 is identical to thesequence of AD-15838.1. Delivery of siRNA duplexes was done using LNPs.Human Hep3B Duplex 10 nM 0.1 nM STDEV, 10 nM STDEV, 0.1 nM AD-15838.20.09 0.66 0.008 0.030 AD-45856.1 0.32 0.91 0.026 0.032 AD-45857.1 2.461.07 0.140 0.044 AD-45858.1 0.10 0.74 0.010 0.070 AD-45860.1 0.02 0.470.002 0.097 AD-45861.1 0.03 0.68 0.004 0.062 AD-45863.1 1.42 0.95 0.1450.126 AD-45864.1 0.02 0.17 0.002 0.045 AD-45865.1 0.32 0.93 0.022 0.062AD-45866.1 0.10 0.92 0.010 0.041 AD-45867.1 0.04 0.61 0.000 0.048AD-45869.1 0.45 1.08 0.028 0.081 AD-45870.1 0.01 0.10 0.003 0.010AD-45871.1 0.05 0.57 0.006 0.071 AD-45872.1 0.07 0.71 0.007 0.034AD-45873.1 0.02 0.23 0.001 0.011 AD-45874.1 0.08 0.75 0.013 0.049AD-45875.1 0.13 0.82 0.017 0.040 AD-45876.1 0.03 0.54 0.000 0.013AD-45877.1 0.06 0.47 0.002 0.025 AD-45878.1 0.02 0.44 0.002 0.031AD-45879.1 0.03 0.35 0.003 0.023 AD-45880.1 0.49 1.00 0.039 0.088AD-45881.1 0.20 0.90 0.019 0.095 AD-45882.1 0.20 0.95 0.012 0.086AD-45883.1 0.16 0.98 0.011 0.058 AD-45884.1 0.09 0.94 0.003 0.044AD-45885.1 0.22 0.91 0.020 0.145 AD-45886.1 0.04 0.40 0.008 0.080AD-45887.1 0.03 0.35 0.002 0.057 AD-45888.1 0.05 0.80 0.006 0.042AD-45889.1 0.31 0.91 0.013 0.052 AD-45890.1 0.06 0.90 0.001 0.047AD-45891.1 0.06 0.82 0.007 0.034 AD-45892.1 1.01 1.09 0.033 0.211AD-45893.1 0.04 0.58 0.002 0.046 AD-45894.1 0.04 0.59 0.003 0.024AD-45895.1 0.84 1.00 0.047 0.047 AD-45896.1 0.84 0.98 0.032 0.095AD-45897.1 0.36 0.61 0.032 0.053 AD-45898.1 0.98 1.09 0.021 0.117AD-45899.1 0.04 0.59 0.005 0.095 AD-45900.1 0.06 0.80 0.005 0.091AD-45901.1 0.33 0.94 0.025 0.096 AD-45902.1 0.24 1.03 0.010 0.079AD-45903.1 0.74 1.02 0.003 0.092 AD-45904.1 0.39 0.87 0.010 0.010AD-45909.1 0.04 0.73 0.008 0.013 AD-45910.1 1.08 1.01 0.037 0.089AD-45914.1 0.52 0.99 0.018 0.071 AD-45915.1 0.06 0.48 0.004 0.046AD-45919.1 0.67 0.98 0.048 0.064 AD-45920.1 0.61 1.00 0.031 0.038AD-45924.1 0.09 0.67 0.005 0.012 AD-45925.1 0.13 0.90 0.008 0.100AD-45929.1 0.02 0.42 0.001 0.083 AD-45930.1 0.05 0.63 0.005 0.052AD-45934.1 0.04 0.41 0.001 0.062 AD-45935.1 0.08 0.76 0.006 0.058AD-45939.1 0.23 0.82 0.030 0.028 AD-1955.1 0.93 0.93 0.068 0.073AD-1955.1 0.94 1.01 0.028 0.113 AD-1955.1 1.00 1.02 0.032 0.065AD-1955.1 1.15 1.06 0.053 0.019

TABLE 5 Dose response screen results for ANGPTL3 dsRNA sequences Theexperiments were conducted using modified oligonucleotide duplexeslisted in Table 3. The sequence of AD-15838.2 is identical to thesequence of AD-15838.1. Hep3B IC₅₀ 24 hrs 120 hrs IC₅₀ IC₅₀ IC₅₀ I IC₅₀II weighted IC₅₀ I IC₅₀ II weighted Duplex (nM) (nM) (nM) (nM) (nM) (nM)AD-15838.2 0.027 0.006 0.017 0.657 0.937 0.800 AD-45860.1 0.006 0.0020.004 0.045 0.032 0.039 AD-45864.1 0.002 0.001 0.002 0.046 0.042 0.044AD-45870.1 0.002 0.001 0.001 0.011 0.008 0.010 AD-45873.1 0.005 0.0040.005 0.037 0.025 0.031 AD-45876.1 0.032 0.006 0.019 0.269 0.045 0.156AD-45877.1 0.018 0.012 0.015 1.660 0.538 1.091 AD-45878.1 0.023 0.0150.019 0.252 0.131 0.190 AD-45879.1 0.002 0.003 0.003 0.023 0.029 0.026AD-45886.1 0.004 0.004 0.004 0.030 0.018 0.025 AD-45887.1 0.010 0.0090.010 0.058 0.059 0.059 AD-45915.1 0.016 0.015 0.015 0.110 0.056 0.083AD-45929.1 0.023 0.008 0.016 0.227 0.025 0.124 AD-45934.1 0.006 0.0060.006 0.110 0.045 0.077

TABLE 6 Results of cell viability screens using modified ANGPTL3 dsRNAsequences The experiments were conducted using modified oligonucleotideduplexes listed in Table 3. The sequence of AD-15838.2 is identical tothe sequence of AD- 15838.1. Viability data is expressed as % viablerelative to mock treated cells. Ave Ave Ave Ave Ave SD SD SD SD SDTarget Duplex 10 nM 1 nM 500 pM 100 pM 50 pM 10 nM 1 nM 500 pM 100 pM 50pM HeLa day 3 ANGPTL3 AD-15838.2 37.34 58.67 70.92 89.86 94.98 9.4512.28 15.06 22.37 18.23 ANGPTL3 AD-15838.2 29.13 48.99 63.18 79.21 94.471.62 5.56 4.34 11.15 11.31 ANGPTL3 AD-45860.1 67.10 75.49 77.93 86.5790.51 6.99 12.93 6.39 6.97 3.57 ANGPTL3 AD-45864.1 99.13 96.95 86.7789.20 84.36 7.90 7.22 12.60 4.85 6.87 ANGPTL3 AD-45870.1 82.36 97.0295.33 95.67 92.27 8.07 5.12 7.97 7.05 10.29 ANGPTL3 AD-45873.1 67.9690.01 90.60 94.20 103.63 11.26 22.61 15.92 22.92 16.97 ANGPTL3AD-45876.1 64.00 76.71 80.21 81.71 91.23 6.60 13.94 10.15 10.81 13.89ANGPTL3 AD-45877.1 79.55 77.33 79.98 91.96 93.46 1.66 9.80 8.73 16.6311.41 ANGPTL3 AD-45878.1 81.95 78.22 78.74 87.93 85.03 15.37 22.72 22.5930.84 40.04 ANGPTL3 AD-45878.1 66.83 70.71 82.14 82.80 83.14 17.48 6.496.86 19.92 21.15 ANGPTL3 AD-45879.1 37.56 45.55 59.28 76.35 78.38 3.507.96 19.73 34.33 33.99 ANGPTL3 AD-45886.1 72.75 57.90 64.51 81.92 82.8914.73 12.64 11.78 25.60 23.14 ANGPTL3 AD-45887.1 38.01 53.91 59.31 76.4485.73 0.58 10.81 6.27 11.12 10.92 ANGPTL3 AD-45915.1 48.06 52.17 67.9095.45 100.77 8.13 15.15 29.11 32.49 38.79 ANGPTL3 AD-45929.1 29.27 44.5852.87 76.45 88.03 4.17 9.67 14.49 31.74 28.82 ANGPTL3 AD-45934.1 68.2064.11 76.92 79.57 92.11 15.79 11.25 19.99 26.08 26.30 (+) controlAD-19200 41.09 85.94 95.13 101.29 96.60 9.99 25.31 24.56 32.26 26.35 (+)control AD-19200 23.99 72.76 86.51 108.10 111.13 5.35 34.52 29.24 35.9931.88 (−) control AD-1955 89.65 99.87 94.59 104.04 105.10 4.57 5.94 4.195.78 7.46 (−) control AD-1955 104.74 99.78 105.79 109.19 108.08 10.947.74 11.12 7.91 10.30 (−) control mock 100.00 6.92 (−) control mock100.00 9.85 (+) control PLK 10.66 26.65 46.16 92.42 98.78 1.70 8.6513.47 22.99 23.48 (+) control PLK 10.74 11.41 17.33 61.02 86.59 3.392.61 1.49 27.42 37.31 HeLa day 6 ANGPTL3 AD-15838.2 47.94 80.97 90.4494.37 96.10 29.05 25.12 13.62 8.88 4.72 ANGPTL3 AD-15838.2 40.32 83.8089.88 95.94 98.27 22.47 16.51 10.03 3.83 4.19 ANGPTL3 AD-45860.1 57.3884.84 88.90 96.74 94.03 24.55 17.35 9.67 3.17 6.58 ANGPTL3 AD-45864.198.65 100.87 101.13 96.86 98.24 4.35 1.91 2.22 3.41 1.80 ANGPTL3AD-45870.1 92.69 98.71 98.49 100.07 99.28 3.94 2.67 2.36 1.19 2.65ANGPTL3 AD-45873.1 91.78 97.38 98.81 97.57 96.22 12.47 6.26 4.08 6.228.64 ANGPTL3 AD-45876.1 63.54 85.68 92.13 96.48 95.97 14.74 16.50 10.035.81 7.51 ANGPTL3 AD-45877.1 94.17 93.21 96.39 96.70 96.98 7.12 8.004.58 3.05 6.15 ANGPTL3 AD-45878.1 66.46 85.75 89.73 94.60 96.59 8.207.41 5.27 3.21 3.91 ANGPTL3 AD-45878.1 70.80 89.30 92.54 96.60 95.095.18 2.13 1.61 0.50 4.15 ANGPTL3 AD-45879.1 8.29 48.25 73.54 87.47 92.194.66 20.05 16.04 9.06 7.90 ANGPTL3 AD-45886.1 23.69 60.65 78.49 93.4194.15 8.19 13.90 7.15 3.35 4.06 ANGPTL3 AD-45887.1 7.24 26.03 57.6895.99 98.80 3.07 13.10 14.94 1.40 2.54 ANGPTL3 AD-45915.1 10.38 58.3885.69 97.24 99.76 6.83 15.66 8.39 1.33 4.15 ANGPTL3 AD-45929.1 11.7336.67 51.90 76.71 85.08 4.80 14.19 15.34 12.37 10.60 ANGPTL3 AD-45934.173.57 88.48 92.94 91.50 95.97 5.36 2.96 5.50 5.44 4.39 (+) controlAD-19200 63.58 90.14 95.44 94.65 93.28 34.11 14.32 8.78 10.90 12.13 (+)control AD-19200 16.05 78.65 85.78 93.09 96.22 9.77 15.57 19.50 13.3410.96 (−) control AD-1955 93.52 97.36 97.90 99.65 100.07 5.02 1.78 0.840.58 1.14 (−) control AD-1955 75.39 93.61 97.79 99.60 100.96 8.37 2.502.27 2.68 3.16 (−) control mock 100.00 1.32 (−) control mock 100.00 3.35(+) control PLK 3.68 55.22 63.00 89.39 95.33 1.42 30.96 33.97 15.85 8.54(+) control PLK 2.69 3.74 9.74 67.07 82.96 0.15 0.96 3.60 22.70 19.34Hep3B day 3 ANGPTL3 AD-15838.2 35.33 61.00 68.79 82.74 90.41 2.41 6.214.21 2.61 7.07 ANGPTL3 AD-15838.2 35.34 61.04 72.14 89.71 106.88 1.492.61 7.37 6.48 7.13 ANGPTL3 AD-45860.1 17.79 39.25 60.57 94.28 99.851.07 3.51 3.57 13.09 16.41 ANGPTL3 AD-45864.1 80.35 88.19 87.01 89.3992.09 6.93 6.98 9.42 7.41 17.05 ANGPTL3 AD-45870.1 75.00 93.30 96.64106.29 99.08 7.10 12.24 4.01 5.95 9.64 ANGPTL3 AD-45873.1 42.68 78.4582.26 97.11 96.58 5.17 5.04 8.31 12.11 11.33 ANGPTL3 AD-45876.1 31.3755.00 70.69 93.49 91.00 4.39 6.09 5.47 15.11 6.38 ANGPTL3 AD-45877.174.45 94.60 96.70 103.77 106.75 3.27 2.44 3.45 6.10 7.40 ANGPTL3AD-45878.1 50.22 69.65 80.49 92.77 97.37 2.51 14.94 10.44 8.21 5.30ANGPTL3 AD-45878.1 44.85 65.39 75.67 92.83 109.67 10.10 7.76 8.56 7.784.97 ANGPTL3 AD-45879.1 23.73 60.81 84.59 95.72 108.68 6.43 21.36 19.6213.69 5.95 ANGPTL3 AD-45886.1 27.19 55.35 64.97 100.18 102.09 0.97 6.6511.46 6.91 4.08 ANGPTL3 AD-45887.1 41.70 97.18 101.91 111.27 105.18 9.266.81 7.36 1.72 2.23 ANGPTL3 AD-45915.1 45.10 66.31 82.22 97.97 103.306.91 11.84 14.79 6.54 2.48 ANGPTL3 AD-45929.1 48.58 79.14 89.96 95.00101.37 10.40 10.29 10.52 18.24 10.53 ANGPTL3 AD-45934.1 80.15 102.93112.82 114.16 113.98 5.28 0.62 4.19 0.75 3.99 (+) control AD-19200 14.7955.23 72.90 89.64 94.30 2.17 5.42 7.19 10.28 16.39 (+) control AD-1920022.76 92.02 101.56 106.68 113.09 6.61 18.99 7.41 9.83 10.64 (−) controlAD-1955 77.77 81.25 82.23 88.21 95.02 2.83 5.40 5.08 5.42 6.63 (−)control AD-1955 80.42 86.70 90.23 93.46 97.04 10.53 5.70 8.14 3.27 3.45(−) control mock 100.00 5.77 (−) control mock 100.00 9.79 (+) controlPLK 10.91 12.89 14.31 23.87 50.93 0.17 0.87 1.64 1.13 7.80 (+) controlPLK 13.19 16.12 22.89 55.03 94.35 0.78 0.88 8.36 18.88 9.85 Hep3B day 6ANGPTL3 AD-15838.2 78.88 89.58 93.08 91.10 100.66 11.60 9.15 12.04 10.515.87 ANGPTL3 AD-15838.2 81.17 85.91 87.27 103.95 103.59 7.75 3.29 8.077.93 9.82 ANGPTL3 AD-45860.1 84.11 87.77 93.22 99.15 96.75 14.22 13.3620.98 13.15 17.62 ANGPTL3 AD-45864.1 99.27 111.82 106.28 99.15 97.557.77 16.31 14.24 15.40 9.18 ANGPTL3 AD-45870.1 95.49 109.60 104.16104.65 106.76 11.92 12.98 9.25 10.29 19.12 ANGPTL3 AD-45873.1 71.4590.62 93.44 102.07 107.72 4.71 4.40 15.02 11.96 10.16 ANGPTL3 AD-45876.176.92 82.09 89.44 95.27 105.41 9.39 13.55 7.93 9.77 10.42 ANGPTL3AD-45877.1 82.98 98.05 95.07 103.55 104.14 11.22 13.45 1.27 8.88 6.49ANGPTL3 AD-45878.1 75.14 82.48 89.68 92.71 95.72 8.65 10.07 10.77 12.4415.04 ANGPTL3 AD-45878.1 65.90 77.37 78.33 84.54 99.49 10.21 13.22 9.9511.65 11.17 ANGPTL3 AD-45879.1 86.42 89.45 101.50 97.30 100.66 10.5910.12 19.77 13.19 9.54 ANGPTL3 AD-45886.1 91.15 79.31 80.76 86.52 94.0412.89 11.88 5.38 4.92 6.80 ANGPTL3 AD-45887.1 91.67 103.38 107.88 100.05102.05 10.80 14.84 19.18 13.72 18.00 ANGPTL3 AD-45915.1 81.97 85.9191.81 94.95 102.13 18.49 19.30 7.19 12.72 16.64 ANGPTL3 AD-45929.1 61.9279.39 87.28 88.09 96.00 6.80 10.76 5.80 10.68 16.66 ANGPTL3 AD-45934.185.84 89.66 97.67 99.91 102.54 12.39 14.25 4.74 9.51 4.28 (+) controlAD-19200 50.48 65.62 79.67 98.61 96.87 4.60 4.64 7.20 5.08 7.37 (+)control AD-19200 52.01 75.89 92.59 101.47 99.66 4.35 20.87 13.57 6.5011.76 (−) control AD-1955 91.77 95.87 93.06 95.10 97.52 8.87 3.46 1.462.00 3.84 (−) control AD-1955 93.65 94.41 89.42 100.59 103.91 9.91 14.906.80 11.99 10.31 (−) control mock 100.00 5.10 (−) control mock 100.007.35 (+) control PLK 36.43 37.75 40.19 55.25 64.59 3.44 2.75 3.65 5.335.02 (+) control PLK 38.70 43.68 50.32 75.17 89.62 3.40 3.85 8.10 10.5410.69

TABLE 7Unmodified sense and antisense strand sequences of ANGPTL3 GalNac-conjugated dsRNAsSense Sequence (SEQ ID NOS 264-448, Antisense Sequence respectively,Position (SEQ ID NOS 449-633, Duplex Sense in order of in Antisenserespectively, in Position in ID Name appearance) NM_014495.2 Nameorder of appearance) NM _014495.2 AD-53063.1 A-108558.1AAAGACAACAAACAUUAUAUUx 1066-1086 A-108559.1 AAUAUAAUGUUUGUUGUCUUUCC1064-1086 AD-52965.1 A-108310.1 ACAAUUAAGCUCCUUCUUUUUx   58-78A-108311.1 AAAAAGAAGGAGCUUAAUUGUGA   56-78 AD-53030.1 A-108410.1UGUCACUUGAACUCAACUCAAx  398-418 A-108411.1 UUGAGUUGAGUUCAAGUGACAUA 396-418 AD-52953.1 A-108306.1 UCACAAUUAAGCUCCUUCUUUx   56-76 A-108307.1AAAGAAGGAGCUUAAUUGUGAAC   54-76 AD-53001.1 A-108416.1CUUGAACUCAACUCAAAACUUx  403-423 A-108417.1 AAGUUUUGAGUUGAGUUCAAGUG 401-423 AD-53080.1 A-108548.1 CUCCAUAGUGAAGCAAUCUAAx 1014-1034A-108549.1 UUAGAUUGCUUCACUAUGGAGUA 1012-1034 AD-52971.1 A-108312.1CAAUUAAGCUCCUUCUUUUUAx   59-79 A-108313.1 UAAAAAGAAGGAGCUUAAUUGUG  57-79 AD-53071.1 A-108498.1 ACCCAGCAACUCUCAAGUUUUx  840-860 A-108499.1AAAACUUGAGAGUUGCUGGGUCU  838-860 AD-53024.1 A-108408.1GAAUAUGUCACUUGAACUCAAx  393-413 A-108409.1 UUGAGUUCAAGUGACAUAUUCUU 391-413 AD-52977.1 A-108314.1 AAUUAAGCUCCUUCUUUUUAUx   60-80 A-108315.1AUAAAAAGAAGGAGCUUAAUUGU   58-80 AD-53064.1 A-108574.1CAUUAUAUUGAAUAUUCUUUUx 1078-1098 A-108575.1 AAAAGAAUAUUCAAUAUAAUGUU1076-1098 AD-53033.1 A-108458.1 ACUAACUAACUUAAUUCAAAAx  483-503A-108459.1 UUUUGAAUUAAGUUAGUUAGUUG  481-503 AD-52954.1 A-108322.1UUAUUGUUCCUCUAGUUAUUUx   77-97 A-108323.1 AAAUAACUAGAGGAACAAUAAAA  75-97 AD-53098.1 A-108554.1 CAUAGUGAAGCAAUCUAAUUAx 1017-1037A-108555.1 UAAUUAGAUUGCUUCACUAUGGA 1015-1037 AD-53092.1 A-108552.1CCAUAGUGAAGCAAUCUAAUUx 1016-1036 A-108553.1 AAUUAGAUUGCUUCACUAUGGAG1014-1036 AD-53073.1 A-108530.1 GAUCACAAAACUUCAAUGAAAx  923-943A-108531.1 UUUCAUUGAAGUUUUGUGAUCCA  921-943 AD-53132.1 A-108628.1AUGGAAGGUUAUACUCUAUAAx 1364-1384 A-108629.1 UUAUAGAGUAUAACCUUCCAUUU1362-1384 AD-53086.1 A-108550.1 UCCAUAGUGAAGCAAUCUAAUx 1015-1035A-108551.1 AUUAGAUUGCUUCACUAUGGAGU 1013-1035 AD-52961.1 A-108340.1CUAUGUUAGACGAUGUAAAAAx  164-184 A-108341.1 UUUUUACAUCGUCUAACAUAGCA 162-184 AD-52983.1 A-108316.1 AUUAAGCUCCUUCUUUUUAUUx   61-81 A-108317.1AAUAAAAAGAAGGAGCUUAAUUG   59-81 AD-53027.1 A-108456.1AACUAACUAACUUAAUUCAAAx  482-502 A-108457.1 UUUGAAUUAAGUUAGUUAGUUGC 480-502 AD-52986.1 A-108364.1 GGCCAAAUUAAUGACAUAUUUx  247-267A-108365.1 AAAUAUGUCAUUAAUUUGGCCCU  245-267 AD-52989.1 A-108318.1UUUUAUUGUUCCUCUAGUUAUx   75-95 A-108319.1 AUAACUAGAGGAACAAUAAAAAG  73-95 AD-52981.1 A-108378.1 ACAUAUUUGAUCAGUCUUUUUx  278-298 A-108379.1AAAAAGACUGAUCAAAUAUGUUG  276-298 AD-53077.1 A-108500.1CCCAGCAACUCUCAAGUUUUUx  841-861 A-108501.1 AAAAACUUGAGAGUUGCUGGGUC 839-861 AD-53095.1 A-108506.1 CAGGUAGUCCAUGGACAUUAAx  884-904A-108507.1 UUAAUGUCCAUGGACUACCUGAU  882-904 AD-52970.1 A-108390.1ACUGAGAAGAACUACAUAUAAx  345-365 A-108391.1 UUAUAUGUAGUUCUUCUCAGUUC 343-365 AD-53015.1 A-108452.1 GAGCAACUAACUAACUUAAUUx  478-498A-108453.1 AAUUAAGUUAGUUAGUUGCUCUU  476-498 AD-53147.1 A-108618.1AACAACCUAAAUGGUAAAUAUx 1282-1302 A-108619.1 AUAUUUACCAUUUAGGUUGUUUU1280-1302 AD-53103.1 A-108540.1 CCUAGAGAAGAUAUACUCCAUx  999-1019A-108541.1 AUGGAGUAUAUCUUCUCUAGGCC  997-1019 AD-52969.1 A-108374.1CAACAUAUUUGAUCAGUCUUUx  276-296 A-108375.1 AAAGACUGAUCAAAUAUGUUGAG 274-296 AD-53075.1 A-108562.1 ACAACAAACAUUAUAUUGAAUx 1070-1090A-108563.1 AUUCAAUAUAAUGUUUGUUGUCU 1068-1090 AD-52994.1 A-108398.1ACAUAUAAACUACAAGUCAAAx  358-378 A-108399.1 UUUGACUUGUAGUUUAUAUGUAG 356-378 AD-52960.1 A-108324.1 CUAGUUAUUUCCUCCAGAAUUx   88-108A-108325.1 AAUUCUGGAGGAAAUAACUAGAG   86-108 AD-53003.1 A-108448.1AAGAGCAACUAACUAACUUAAx  476-496 A-108449.1 UUAAGUUAGUUAGUUGCUCUUCU 474-496 AD-52995.1 A-108320.1 UUUAUUGUUCCUCUAGUUAUUx   76-96 A-108321.1AAUAACUAGAGGAACAAUAAAAA   74-96 AD-53037.1 A-108428.1CUCCUAGAAGAAAAAAUUCUAx  430-450 A-108429.1 UAGAAUUUUUUCUUCUAGGAGGC 428-450 AD-53087.1 A-108566.1 AACAAACAUUAUAUUGAAUAUx 1072-1092A-108567.1 AUAUUCAAUAUAAUGUUUGUUGU 1070-1092 AD-53076.1 A-108578.1GGAAAUCACGAAACCAACUAUx 1105-1125 A-108579.1 AUAGUUGGUUUCGUGAUUUCCCA1103-1125 AD-52975.1 A-108376.1 AACAUAUUUGAUCAGUCUUUUx  277-297A-108377.1 AAAAGACUGAUCAAAUAUGUUGA  275-297 AD-53138.1 A-108630.1UGGAAGGUUAUACUCUAUAAAx 1365-1385 A-108631.1 UUUAUAGAGUAUAACCUUCCAUU1363-1385 AD-53091.1 A-108536.1 GGAGAACUACAAAUAUGGUUUx  948-968A-108537.1 AAACCAUAUUUGUAGUUCUCCCA  946-968 AD-53124.1 A-108594.1GAAAACAAAGAUUUGGUGUUUx 1174-1194 A-108595.1 AAACACCAAAUCUUUGUUUUCCG1172-1194 AD-53125.1 A-108610.1 AGUGUGGAGAAAACAACCUAAx 1271-1291A-108611.1 UUAGGUUGUUUUCUCCACACUCA 1269-1291 AD-53036.1 A-108412.1GUCACUUGAACUCAACUCAAAx  399-419 A-108413.1 UUUGAGUUGAGUUCAAGUGACAU 397-419 AD-53061.1 A-108526.1 GAUGGAUCACAAAACUUCAAUx  919-939A-108527.1 AUUGAAGUUUUGUGAUCCAUCUA  917-939 AD-53093.1 A-108568.1ACAAACAUUAUAUUGAAUAUUx 1073-1093 A-108569.1 AAUAUUCAAUAUAAUGUUUGUUG1071-1093 AD-53137.1 A-108614.1 UGUGGAGAAAACAACCUAAAUx 1273-1293A-108615.1 AUUUAGGUUGUUUUCUCCACACU 1271-1293 AD-52999.1 A-108384.1AUCAGUCUUUUUAUGAUCUAUx  287-307 A-108385.1 AUAGAUCAUAAAAAGACUGAUCA 285-307 AD-53069.1 A-108560.1 GACAACAAACAUUAUAUUGAAx 1069-1089A-108561.1 UUCAAUAUAAUGUUUGUUGUCUU 1067-1089 AD-53034.1 A-108474.1CAACAGCAUAGUCAAAUAAAAx  622-642 A-108475.1 UUUUAUUUGACUAUGCUGUUGGU 620-642 AD-52976.1 A-108392.1 CUGAGAAGAACUACAUAUAAAx  346-366A-108393.1 UUUAUAUGUAGUUCUUCUCAGUU  344-366 AD-52996.1 A-108336.1UGCUAUGUUAGACGAUGUAAAx  162-182 A-108337.1 UUUACAUCGUCUAACAUAGCAAA 160-182 AD-53029.1 A-108488.1 AACCCACAGAAAUUUCUCUAUx  680-700A-108489.1 AUAGAGAAAUUUCUGUGGGUUCU  678-700 AD-53020.1 A-108438.1CUUCAACAAAAAGUGAAAUAUx  451-471 A-108439.1 AUAUUUCACUUUUUGUUGAAGUA 449-471 AD-53042.1 A-108414.1 UCACUUGAACUCAACUCAAAAx  400-420A-108415.1 UUUUGAGUUGAGUUCAAGUGACA  398-420 AD-53011.1 A-108482.1CAUAGUCAAAUAAAAGAAAUAx  628-648 A-108483.1 UAUUUCUUUUAUUUGACUAUGCU 626-648 AD-52957.1 A-108370.1 CAAAAACUCAACAUAUUUGAUx  268-288A-108371.1 AUCAAAUAUGUUGAGUUUUUGAA  266-288 AD-53008.1 A-108434.1UACUUCAACAAAAAGUGAAAUx  449-469 A-108435.1 AUUUCACUUUUUGUUGAAGUAGA 447-469 AD-53065.1 A-108496.1 GACCCAGCAACUCUCAAGUUUx  839-859A-108497.1 AAACUUGAGAGUUGCUGGGUCUG  837-859 AD-53115.1 A-108638.1UUGAAUGAACUGAGGCAAAUUx 1427-1447 A-108639.1 AAUUUGCCUCAGUUCAUUCAAAG1425-1447 AD-53012.1 A-108404.1 UAUAAACUACAAGUCAAAAAUx  361-381A-108405.1 AUUUUUGACUUGUAGUUUAUAUG  359-381 AD-53004.1 A-108464.1AAACAAGAUAAUAGCAUCAAAx  559-579 A-108465.1 UUUGAUGCUAUUAUCUUGUUUUU 557-579 AD-53021.1 A-108454.1 CAACUAACUAACUUAAUUCAAx  481-501A-108455.1 UUGAAUUAAGUUAGUUAGUUGCU  479-501 AD-52955.1 A-108338.1GCUAUGUUAGACGAUGUAAAAx  163-183 A-108339.1 UUUUACAUCGUCUAACAUAGCAA 161-183 AD-53119.1 A-108608.1 ACUUGGGAUCACAAAGCAAAAx 1198-1218A-108609.1 UUUUGCUUUGUGAUCCCAAGUAG 1196-1218 AD-52990.1 A-108334.1UUGCUAUGUUAGACGAUGUAAx  161-181 A-108335.1 UUACAUCGUCUAACAUAGCAAAU 159-181 AD-52964.1 A-108388.1 AACUGAGAAGAACUACAUAUAx  344-364A-108389.1 UAUAUGUAGUUCUUCUCAGUUCC  342-364 AD-52973.1 A-108344.1GAUGUAAAAAUUUUAGCCAAUx  175-195 A-108345.1 AUUGGCUAAAAUUUUUACAUCGU 173-195 AD-53074.1 A-108546.1 ACUCCAUAGUGAAGCAAUCUAx 1013-1033A-108547.1 UAGAUUGCUUCACUAUGGAGUAU 1011-1033 AD-53026.1 A-108440.1UUCAACAAAAAGUGAAAUAUUx  452-472 A-108441.1 AAUAUUUCACUUUUUGUUGAAGU 450-472 AD-53062.1 A-108542.1 CUAGAGAAGAUAUACUCCAUAx 1000-1020A-108543.1 UAUGGAGUAUAUCUUCUCUAGGC  998-1020 AD-53114.1 A-108622.1CAACCUAAAUGGUAAAUAUAAx 1284-1304 A-108623.1 UUAUAUUUACCAUUUAGGUUGUU1282-1304 AD-53082.1 A-108580.1 GAAAUCACGAAACCAACUAUAx 1106-1126A-108581.1 UAUAGUUGGUUUCGUGAUUUCCC 1104-1126 AD-53035.1 A-108490.1CCACAGAAAUUUCUCUAUCUUx  683-703 A-108491.1 AAGAUAGAGAAAUUUCUGUGGGU 681-703 AD-52978.1 A-108330.1 AAAUCAAGAUUUGCUAUGUUAx  151-171A-108331.1 UAACAUAGCAAAUCUUGAUUUUG  149-171 AD-53084.1 A-108518.1ACAUUAAUUCAACAUCGAAUAx  898-918 A-108519.1 UAUUCGAUGUUGAAUUAAUGUCC 896-918 AD-52972.1 A-108328.1 CCAGAGCCAAAAUCAAGAUUUx  142-162A-108329.1 AAAUCUUGAUUUUGGCUCUGGAG  140-162 AD-53002.1 A-108432.1CUACUUCAACAAAAAGUGAAAx  448-468 A-108433.1 UUUCACUUUUUGUUGAAGUAGAA 446-468 AD-53078.1 A-108516.1 GACAUUAAUUCAACAUCGAAUx  897-917A-108517.1 AUUCGAUGUUGAAUUAAUGUCCA  895-917 AD-53072.1 A-108514.1GGACAUUAAUUCAACAUCGAAx  896-916 A-108515.1 UUCGAUGUUGAAUUAAUGUCCAU 894-916 AD-53005.1 A-108480.1 GCAUAGUCAAAUAAAAGAAAUx  627-647A-108481.1 AUUUCUUUUAUUUGACUAUGCUG  625-647 AD-53083.1 A-108502.1CUCUCAAGUUUUUCAUGUCUAx  849-869 A-108503.1 UAGACAUGAAAAACUUGAGAGUU 847-869 AD-53102.1 A-108524.1 AUCGAAUAGAUGGAUCACAAAx  911-931A-108525.1 UUUGUGAUCCAUCUAUUCGAUGU  909-931 AD-53105.1 A-108572.1ACAUUAUAUUGAAUAUUCUUUx 1077-1097 A-108573.1 AAAGAAUAUUCAAUAUAAUGUUU1075-1097 AD-53090.1 A-108520.1 UUAAUUCAACAUCGAAUAGAUx  901-921A-108521.1 AUCUAUUCGAUGUUGAAUUAAUG  899-921 AD-53010.1 A-108466.1GAUAAUAGCAUCAAAGACCUUx  565-585 A-108467.1 AAGGUCUUUGAUGCUAUUAUCUU 563-585 AD-52998.1 A-108368.1 UGACAUAUUUCAAAAACUCAAx  258-278A-108369.1 UUGAGUUUUUGAAAUAUGUCAUU  256-278 AD-52992.1 A-108366.1AAAUUAAUGACAUAUUUCAAAx  251-271 A-108367.1 UUUGAAAUAUGUCAUUAAUUUGG 249-271 AD-53068.1 A-108544.1 GAAGAUAUACUCCAUAGUGAAx 1005-1025A-108545.1 UUCACUAUGGAGUAUAUCUUCUC 1003-1025 AD-53032.1 A-108442.1AAUAUUUAGAAGAGCAACUAAx  467-487 A-108443.1 UUAGUUGCUCUUCUAAAUAUUUC 465-487 AD-52967.1 A-108342.1 CGAUGUAAAAAUUUUAGCCAAx  174-194A-108343.1 UUGGCUAAAAUUUUUACAUCGUC  172-194 AD-53096.1 A-108522.1UUCAACAUCGAAUAGAUGGAUx  905-925 A-108523.1 AUCCAUCUAUUCGAUGUUGAAUU 903-925 AD-53131.1 A-108612.1 GUGUGGAGAAAACAACCUAAAx 1272-1292A-108613.1 UUUAGGUUGUUUUCUCCACACUC 1270-1292 AD-52963.1 A-108372.1UCAACAUAUUUGAUCAGUCUUx  275-295 A-108373.1 AAGACUGAUCAAAUAUGUUGAGU 273-295 AD-53089.1 A-108504.1 UCAGGUAGUCCAUGGACAUUAx  883-903A-108505.1 UAAUGUCCAUGGACUACCUGAUA  881-903 AD-53044.1 A-108446.1UUUAGAAGAGCAACUAACUAAx  471-491 A-108447.1 UUAGUUAGUUGCUCUUCUAAAUA 469-491 AD-52988.1 A-108396.1 UACAUAUAAACUACAAGUCAAx  357-377A-108397.1 UUGACUUGUAGUUUAUAUGUAGU  355-377 AD-53067.1 A-108528.1GGAUCACAAAACUUCAAUGAAx  922-942 A-108529.1 UUCAUUGAAGUUUUGUGAUCCAU 920-942 AD-53009.1 A-108450.1 AGAGCAACUAACUAACUUAAUx  477-497A-108451.1 AUUAAGUUAGUUAGUUGCUCUUC  475-497 AD-53022.1 A-108470.1ACCAACAGCAUAGUCAAAUAAx  620-640 A-108471.1 UUAUUUGACUAUGCUGUUGGUUU 618-640 AD-53016.1 A-108468.1 AACCAACAGCAUAGUCAAAUAx  619-639A-108469.1 UAUUUGACUAUGCUGUUGGUUUA  617-639 AD-53007.1 A-108418.1GAACUCAACUCAAAACUUGAAx  406-426 A-108419.1 UUCAAGUUUUGAGUUGAGUUCAA 404-426 AD-53148.1 A-108634.1 UACUCUAUAAAAUCAACCAAAx 1375-1395A-108635.1 UUUGGUUGAUUUUAUAGAGUAUA 1373-1395 AD-53040.1 A-108476.1CAGCAUAGUCAAAUAAAAGAAx  625-645 A-108477.1 UUCUUUUAUUUGACUAUGCUGUU 623-645 AD-53041.1 A-108492.1 GAAAUAAGAAAUGUAAAACAUx  748-768A-108493.1 AUGUUUUACAUUUCUUAUUUCAU  746-768 AD-53039.1 A-108460.1CUAACUAACUUAAUUCAAAAUx  484-504 A-108461.1 AUUUUGAAUUAAGUUAGUUAGUU 482-504 AD-53139.1 A-108646.1 AUGAACUGAGGCAAAUUUAAAx 1431-1451A-108647.1 UUUAAAUUUGCCUCAGUUCAUUC 1429-1451 AD-53144.1 A-108648.1UGAACUGAGGCAAAUUUAAAAx 1432-1452 A-108649.1 UUUUAAAUUUGCCUCAGUUCAUU1430-1452 AD-53142.1 A-108616.1 AAACAACCUAAAUGGUAAAUAx 1281-1301A-108617.1 UAUUUACCAUUUAGGUUGUUUUC 1279-1301 AD-53108.1 A-108620.1ACAACCUAAAUGGUAAAUAUAx 1283-1303 A-108621.1 UAUAUUUACCAUUUAGGUUGUUU1281-1303 AD-53079.1 A-108532.1 AACGUGGGAGAACUACAAAUAx  942-962A-108533.1 UAUUUGUAGUUCUCCCACGUUUC  940-962 AD-53133.1 A-108644.1AAUGAACUGAGGCAAAUUUAAx 1430-1450 A-108645.1 UUAAAUUUGCCUCAGUUCAUUCA1428-1450 AD-53104.1 A-108556.1 GUUGGAAGACUGGAAAGACAAx 1053-1073A-108557.1 UUGUCUUUCCAGUCUUCCAACUC 1051-1073 AD-53088.1 A-108582.1UGGCAAUGUCCCCAAUGCAAUx 1149-1169 A-108583.1 AUUGCAUUGGGGACAUUGCCAGU1147-1169 AD-53101.1 A-108508.1 GGUAGUCCAUGGACAUUAAUUx  886-906A-108509.1 AAUUAAUGUCCAUGGACUACCUG  884-906 AD-53000.1 A-108400.1CAUAUAAACUACAAGUCAAAAx  359-379 A-108401.1 UUUUGACUUGUAGUUUAUAUGUA 357-379 AD-53112.1 A-108590.1 AAUCCCGGAAAACAAAGAUUUx 1167-1187A-108591.1 AAAUCUUUGUUUUCCGGGAUUGC 1165-1187 AD-53107.1 A-108604.1CUACUUGGGAUCACAAAGCAAx 1196-1216 A-108605.1 UUGCUUUGUGAUCCCAAGUAGAA1194-1216 AD-53121.1 A-108640.1 UGAAUGAACUGAGGCAAAUUUx 1428-1448A-108641.1 AAAUUUGCCUCAGUUCAUUCAAA 1426-1448 AD-53046.1 A-108478.1AGCAUAGUCAAAUAAAAGAAAx  626-646 A-108479.1 UUUCUUUUAUUUGACUAUGCUGU 624-646 AD-53038.1 A-108444.1 AUUUAGAAGAGCAACUAACUAx  470-490A-108445.1 UAGUUAGUUGCUCUUCUAAAUAU  468-490 AD-53140.1 A-108662.1AGGCAAAUUUAAAAGGCAAUAx 1439-1459 A-108663.1 UAUUGCCUUUUAAAUUUGCCUCA1437-1459 AD-52987.1 A-108380.1 CAUAUUUGAUCAGUCUUUUUAx  279-299A-108381.1 UAAAAAGACUGAUCAAAUAUGUU  277-299 AD-53130.1 A-108596.1AAAACAAAGAUUUGGUGUUUUx 1175-1195 A-108597.1 AAAACACCAAAUCUUUGUUUUCC1173-1195 AD-53106.1 A-108588.1 CAAUCCCGGAAAACAAAGAUUx 1166-1186A-108589.1 AAUCUUUGUUUUCCGGGAUUGCA 1164-1186 AD-53081.1 A-108564.1CAACAAACAUUAUAUUGAAUAx 1071-1091 A-108565.1 UAUUCAAUAUAAUGUUUGUUGUC1069-1091 AD-53118.1 A-108592.1 GGAAAACAAAGAUUUGGUGUUx 1173-1193A-108593.1 AACACCAAAUCUUUGUUUUCCGG 1171-1193 AD-53136.1 A-108598.1ACAAAGAUUUGGUGUUUUCUAx 1178-1198 A-108599.1 UAGAAAACACCAAAUCUUUGUUU1176-1198 AD-53127.1 A-108642.1 GAAUGAACUGAGGCAAAUUUAx 1429-1449A-108643.1 UAAAUUUGCCUCAGUUCAUUCAA 1427-1449 AD-53066.1 A-108512.1CCAUGGACAUUAAUUCAACAUx  892-912 A-108513.1 AUGUUGAAUUAAUGUCCAUGGAC 890-912 AD-53013.1 A-108420.1 AACUCAACUCAAAACUUGAAAx  407-427A-108421.1 UUUCAAGUUUUGAGUUGAGUUCA  405-427 AD-52991.1 A-108350.1CAGUUGGGACAUGGUCUUAAAx  205-225 A-108351.1 UUUAAGACCAUGUCCCAACUGAA 203-225 AD-53099.1 A-108570.1 AACAUUAUAUUGAAUAUUCUUx 1076-1096A-108571.1 AAGAAUAUUCAAUAUAAUGUUUG 1074-1096 AD-52958.1 A-108386.1ACCAGUGAAAUCAAAGAAGAAx  316-336 A-108387.1 UUCUUCUUUGAUUUCACUGGUUU 314-336 AD-53097.1 A-108538.1 GUUGGGCCUAGAGAAGAUAUAx  993-1013A-108539.1 UAUAUCUUCUCUAGGCCCAACCA  991-1013 AD-52966.1 A-108326.1CUCCAGAGCCAAAAUCAAGAUx  140-160 A-108327.1 AUCUUGAUUUUGGCUCUGGAGAU 138-160 AD-53145.1 A-108664.1 GGCAAAUUUAAAAGGCAAUAAx 1440-1460A-108665.1 UUAUUGCCUUUUAAAUUUGCCUC 1438-1460 AD-53113.1 A-108606.1UACUUGGGAUCACAAAGCAAAx 1197-1217 A-108607.1 UUUGCUUUGUGAUCCCAAGUAGA1195-1217 AD-52993.1 A-108382.1 GAUCAGUCUUUUUAUGAUCUAx  286-306A-108383.1 UAGAUCAUAAAAAGACUGAUCAA  284-306 AD-53031.1 A-108426.1GAAAGCCUCCUAGAAGAAAAAx  424-444 A-108427.1 UUUUUCUUCUAGGAGGCUUUCAA 422-444 AD-53017.1 A-108484.1 AGUCAAAUAAAAGAAAUAGAAx  631-651A-108485.1 UUCUAUUUCUUUUAUUUGACUAU  629-651 AD-53143.1 A-108632.1AUACUCUAUAAAAUCAACCAAx 1374-1394 A-108633.1 UUGGUUGAUUUUAUAGAGUAUAA1372-1394 AD-53149.1 A-108650.1 GAACUGAGGCAAAUUUAAAAAx 1433-1453A-108651.1 UUUUUAAAUUUGCCUCAGUUCAU 1431- 1453_G21A AD-53059.1 A-108494.1AGACCCAGCAACUCUCAAGUUx  838-858 A-108495.1 AACUUGAGAGUUGCUGGGUCUGA 836-858 AD-53006.1 A-108402.1 AUAUAAACUACAAGUCAAAAAx  360-380A-108403.1 UUUUUGACUUGUAGUUUAUAUGU  358-380 AD-53025.1 A-108424.1UGAAAGCCUCCUAGAAGAAAAx  423-443 A-108425.1 UUUUCUUCUAGGAGGCUUUCAAG 421-443 AD-53085.1 A-108534.1 GGGAGAACUACAAAUAUGGUUx  947-967A-108535.1 AACCAUAUUUGUAGUUCUCCCAC  945-967 AD-52984.1 A-108332.1AGAUUUGCUAUGUUAGACGAUx  157-177 A-108333.1 AUCGUCUAACAUAGCAAAUCUUG 155-177 AD-53023.1 A-108486.1 GAACCCACAGAAAUUUCUCUAx  679-699A-108487.1 UAGAGAAAUUUCUGUGGGUUCUU  677-699 AD-53014.1 A-108436.1ACUUCAACAAAAAGUGAAAUAx  450-470 A-108437.1 UAUUUCACUUUUUGUUGAAGUAG 448-470 AD-53060.1 A-108510.1 AGUCCAUGGACAUUAAUUCAAx  889-909A-108511.1 UUGAAUUAAUGUCCAUGGACUAC  887-909 AD-53110.1 A-108652.1AACUGAGGCAAAUUUAAAAGAx 1434-1454 A-108653.1 UCUUUUAAAUUUGCCUCAGUUCA1432- 1454_G21A AD-52980.1 A-108362.1 GGGCCAAAUUAAUGACAUAUUx  246-266A-108363.1 AAUAUGUCAUUAAUUUGGCCCUU  244-266 AD-53109.1 A-108636.1AUCCAUCCAACAGAUUCAGAAx 1402-1422 A-108637.1 UUCUGAAUCUGUUGGAUGGAUCA1400-1422 AD-53141.1 A-108600.1 AAGAUUUGGUGUUUUCUACUUx 1181-1201A-108601.1 AAGUAGAAAACACCAAAUCUUUG 1179-1201 AD-53126.1 A-108626.1GUCUCAAAAUGGAAGGUUAUAx 1356-1376 A-108627.1 UAUAACCUUCCAUUUUGAGACUU1354-1376 AD-53116.1 A-108654.1 ACUGAGGCAAAUUUAAAAGGAx 1435-1455A-108655.1 UCCUUUUAAAUUUGCCUCAGUUC 1433- 1455_C21A AD-52997.1 A-108352.1GGGACAUGGUCUUAAAGACUUx  210-230 A-108353.1 AAGUCUUUAAGACCAUGUCCCAA 208-230 AD-53120.1 A-108624.1 AUGGUAAAUAUAACAAACCAAx 1292-1312A-108625.1 UUGGUUUGUUAUAUUUACCAUUU 1290-1312 AD-53070.1 A-108576.1GGGAAAUCACGAAACCAACUAx 1104-1124 A-108577.1 UAGUUGGUUUCGUGAUUUCCCAA1102-1124 AD-53028.1 A-108472.1 CCAACAGCAUAGUCAAAUAAAx  621-641A-108473.1 UUUAUUUGACUAUGCUGUUGGUU  619-641 AD-53146.1 A-108602.1UUUUCUACUUGGGAUCACAAAx 1192-1212 A-108603.1 UUUGUGAUCCCAAGUAGAAAACA1190-1212 AD-52982.1 A-108394.1 AGAACUACAUAUAAACUACAAx  352-372A-108395.1 UUGUAGUUUAUAUGUAGUUCUUC  350-372 AD-53111.1 A-108668.1AGAGUAUGUGUAAAAAUCUGUx 1915-1935 A-108669.1 ACAGAUUUUUACACAUACUCUGU1913-1935 AD-53045.1 A-108462.1 AAAACAAGAUAAUAGCAUCAAx  558-578A-108463.1 UUGAUGCUAUUAUCUUGUUUUUC  556-578 AD-53123.1 A-108672.1AGUAUGUGUAAAAAUCUGUAAx 1917-1937 A-108673.1 UUACAGAUUUUUACACAUACUCU1915-1937 AD-53018.1 A-108406.1 AGUCAAAAAUGAAGAGGUAAAx  372-392A-108407.1 UUUACCUCUUCAUUUUUGACUUG  370-392 AD-52956.1 A-108354.1GGACAUGGUCUUAAAGACUUUx  211-231 A-108355.1 AAAGUCUUUAAGACCAUGUCCCA 209-231 AD-53134.1 A-108660.1 GAGGCAAAUUUAAAAGGCAAUx 1438-1458A-108661.1 AUUGCCUUUUAAAUUUGCCUCAG 1436-1458 AD-52968.1 A-108358.1GUCUUAAAGACUUUGUCCAUAx  218-238 A-108359.1 UAUGGACAAAGUCUUUAAGACCA 216-238 AD-53122.1 A-108656.1 CUGAGGCAAAUUUAAAAGGCAx 1436-1456A-108657.1 UGCCUUUUAAAUUUGCCUCAGUU 1434-1456 AD-53100.1 A-108586.1GCAAUCCCGGAAAACAAAGAUx 1165-1185 A-108587.1 AUCUUUGUUUUCCGGGAUUGCAU1163-1185 AD-53128.1 A-108658.1 UGAGGCAAAUUUAAAAGGCAAx 1437-1457A-108659.1 UUGCCUUUUAAAUUUGCCUCAGU 1435-1457 AD-53043.1 A-108430.1UCUACUUCAACAAAAAGUGAAx  447-467 A-108431.1 UUCACUUUUUGUUGAAGUAGAAU 445-467 AD-53135.1 A-108676.1 UAUGUGUAAAAAUCUGUAAUAx 1919-1939A-108677.1 UAUUACAGAUUUUUACACAUACU 1917-1939 AD-53094.1 A-108584.1AAUGCAAUCCCGGAAAACAAAx 1162-1182 A-108585.1 UUUGUUUUCCGGGAUUGCAUUGG1160-1182 AD-53019.1 A-108422.1 CUUGAAAGCCUCCUAGAAGAAx  421-441A-108423.1 UUCUUCUAGGAGGCUUUCAAGUU  419-441 AD-53129.1 A-108674.1GUAUGUGUAAAAAUCUGUAAUx 1918-1938 A-108675.1 AUUACAGAUUUUUACACAUACUC1916-1938 AD-53150.1 A-108666.1 CAGAGUAUGUGUAAAAAUCUUx 1914-1934A-108667.1 AAGAUUUUUACACAUACUCUGUG 1912- 1934_G21U AD-53117.1 A-108670.1GAGUAUGUGUAAAAAUCUGUAx 1916-1936 A-108671.1 UACAGAUUUUUACACAUACUCUG1914-1936 AD-52985.1 A-108348.1 UCAGUUGGGACAUGGUCUUAAx  204-224A-108349.1 UUAAGACCAUGUCCCAACUGAAG  202-224 AD-52962.1 A-108356.1GGUCUUAAAGACUUUGUCCAUx  217-237 A-108357.1 AUGGACAAAGUCUUUAAGACCAU 215-237 AD-52974.1 A-108360.1 UCUUAAAGACUUUGUCCAUAAx  219-239A-108361.1 UUAUGGACAAAGUCUUUAAGACC  217-239 AD-52979.1 A-108346.1UUCAGUUGGGACAUGGUCUUAx  203-223 A-108347.1 UAAGACCAUGUCCCAACUGAAGG 201-223 The symbol “x” indicates that the sequence contains a GalNAcconjugate.

TABLE 8 Modified sense and antisense strandsequences of ANGPTL3 GalNac-conjugated dsRNAs Sense Sequence Sense(SEQ ID NOS 634-818, respectively, in Duplex ID OligoNameorder of appearance) AD-53063.1 A-108558.1AfaAfgAfcAfaCfAfAfaCfaUfuAfuAfuUfL96 AD-52965.1 A-108310.1AfcAfaUfuAfaGfCfUfcCfuUfcUfuUfuUfL96 AD-53030.1 A-108410.1UfgUfcAfcUfuGfAfAfcUfcAfaCfuCfaAfL96 AD-52953.1 A-108306.1UfcAfcAfaUfuAfAfGfcUfcCfuUfcUfuUfL96 AD-53001.1 A-108416.1CfuUfgAfaCfuCfAfAfcUfcAfaAfaCfuUfL96 AD-53080.1 A-108548.1CfuCfcAfuAfgUfGfAfaGfcAfaUfcUfaAfL96 AD-52971.1 A-108312.1CfaAfuUfaAfgCfUfCfcUfuCfuUfuUfuAfL96 AD-53071.1 A-108498.1AfcCfcAfgCfaAfCfUfcUfcAfaGfuUfuUfL96 AD-53024.1 A-108408.1GfaAfuAfuGfuCfAfCfuUfgAfaCfuCfaAfL96 AD-52977.1 A-108314.1AfaUfuAfaGfcUfCfCfuUfcUfuUfuUfaUfL96 AD-53064.1 A-108574.1CfaUfuAfuAfuUfGfAfaUfaUfuCfuUfuUfL96 AD-53033.1 A-108458.1AfcUfaAfcUfaAfCfUfuAfaUfuCfaAfaAfL96 AD-52954.1 A-108322.1UfuAfuUfgUfuCfCfUfcUfaGfuUfaUfuUfL96 AD-53098.1 A-108554.1CfaUfaGfuGfaAfGfCfaAfuCfuAfaUfuAfL96 AD-53092.1 A-108552.1CfcAfuAfgUfgAfAfGfcAfaUfcUfaAfuUfL96 AD-53073.1 A-108530.1GfaUfcAfcAfaAfAfCfuUfcAfaUfgAfaAfL96 AD-53132.1 A-108628.1AfuGfgAfaGfgUfUfAfuAfcUfcUfaUfaAfL96 AD-53086.1 A-108550.1UfcCfaUfaGfuGfAfAfgCfaAfuCfuAfaUfL96 AD-52961.1 A-108340.1CfuAfuGfuUfaGfAfCfgAfuGfuAfaAfaAfL96 AD-52983.1 A-108316.1AfuUfaAfgCfuCfCfUfuCfuUfuUfuAfuUfL96 AD-53027.1 A-108456.1AfaCfuAfaCfuAfAfCfuUfaAfuUfcAfaAfL96 AD-52986.1 A-108364.1GfgCfcAfaAfuUfAfAfuGfaCfaUfaUfuUfL96 AD-52989.1 A-108318.1UfuUfuAfuUfgUfUfCfcUfcUfaGfuUfaUfL96 AD-52981.1 A-108378.1AfcAfuAfuUfuGfAfUfcAfgUfcUfuUfuUfL96 AD-53077.1 A-108500.1CfcCfaGfcAfaCfUfCfuCfaAfgUfuUfuUfL96 AD-53095.1 A-108506.1CfaGfgUfaGfuCfCfAfuGfgAfcAfuUfaAfL96 AD-52970.1 A-108390.1AfcUfgAfgAfaGfAfAfcUfaCfaUfaUfaAfL96 AD-53015.1 A-108452.1GfaGfcAfaCfuAfAfCfuAfaCfuUfaAfuUfL96 AD-53147.1 A-108618.1AfaCfaAfcCfuAfAfAfuGfgUfaAfaUfaUfL96 AD-53103.1 A-108540.1CfcUfaGfaGfaAfGfAfuAfuAfcUfcCfaUfL96 AD-52969.1 A-108374.1CfaAfcAfuAfuUfUfGfaUfcAfgUfcUfuUfL96 AD-53075.1 A-108562.1AfcAfaCfaAfaCfAfUfuAfuAfuUfgAfaUfL96 AD-52994.1 A-108398.1AfcAfuAfuAfaAfCfUfaCfaAfgUfcAfaAfL96 AD-52960.1 A-108324.1CfuAfgUfuAfuUfUfCfcUfcCfaGfaAfuUfL96 AD-53003.1 A-108448.1AfaGfaGfcAfaCfUfAfaCfuAfaCfuUfaAfL96 AD-52995.1 A-108320.1UfuUfaUfuGfuUfCfCfuCfuAfgUfuAfuUfL96 AD-53037.1 A-108428.1CfuCfcUfaGfaAfGfAfaAfaAfaUfuCfuAfL96 AD-53087.1 A-108566.1AfaCfaAfaCfaUfUfAfuAfuUfgAfaUfaUfL96 AD-53076.1 A-108578.1GfgAfaAfuCfaCfGfAfaAfcCfaAfcUfaUfL96 AD-52975.1 A-108376.1AfaCfaUfaUfuUfGfAfuCfaGfuCfuUfuUfL96 AD-53138.1 A-108630.1UfgGfaAfgGfuUfAfUfaCfuCfuAfuAfaAfL96 AD-53091.1 A-108536.1GfgAfgAfaCfuAfCfAfaAfuAfuGfgUfuUfL96 AD-53124.1 A-108594.1GfaAfaAfcAfaAfGfAfuUfuGfgUfgUfuUfL96 AD-53125.1 A-108610.1AfgUfgUfgGfaGfAfAfaAfcAfaCfcUfaAfL96 AD-53036.1 A-108412.1GfuCfaCfuUfgAfAfCfuCfaAfcUfcAfaAfL96 AD-53061.1 A-108526.1GfaUfgGfaUfcAfCfAfaAfaCfuUfcAfaUfL96 AD-53093.1 A-108568.1AfcAfaAfcAfuUfAfUfaUfuGfaAfuAfuUfL96 AD-53137.1 A-108614.1UfgUfgGfaGfaAfAfAfcAfaCfcUfaAfaUfL96 AD-52999.1 A-108384.1AfuCfaGfuCfuUfUfUfuAfuGfaUfcUfaUfL96 AD-53069.1 A-108560.1GfaCfaAfcAfaAfCfAfuUfaUfaUfuGfaAfL96 AD-53034.1 A-108474.1CfaAfcAfgCfaUfAfGfuCfaAfaUfaAfaAfL96 AD-52976.1 A-108392.1CfuGfaGfaAfgAfAfCfuAfcAfuAfuAfaAfL96 AD-52996.1 A-108336.1UfgCfuAfuGfuUfAfGfaCfgAfuGfuAfaAfL96 AD-53029.1 A-108488.1AfaCfcCfaCfaGfAfAfaUfuUfcUfcUfaUfL96 AD-53020.1 A-108438.1CfuUfcAfaCfaAfAfAfaGfuGfaAfaUfaUfL96 AD-53042.1 A-108414.1UfcAfcUfuGfaAfCfUfcAfaCfuCfaAfaAfL96 AD-53011.1 A-108482.1CfaUfaGfuCfaAfAfUfaAfaAfgAfaAfuAfL96 AD-52957.1 A-108370.1CfaAfaAfaCfuCfAfAfcAfuAfuUfuGfaUfL96 AD-53008.1 A-108434.1UfaCfuUfcAfaCfAfAfaAfaGfuGfaAfaUfL96 AD-53065.1 A-108496.1GfaCfcCfaGfcAfAfCfuCfuCfaAfgUfuUfL96 AD-53115.1 A-108638.1UfuGfaAfuGfaAfCfUfgAfgGfcAfaAfuUfL96 AD-53012.1 A-108404.1UfaUfaAfaCfuAfCfAfaGfuCfaAfaAfaUfL96 AD-53004.1 A-108464.1AfaAfcAfaGfaUfAfAfuAfgCfaUfcAfaAfL96 AD-53021.1 A-108454.1CfaAfcUfaAfcUfAfAfcUfuAfaUfuCfaAfL96 AD-52955.1 A-108338.1GfcUfaUfgUfuAfGfAfcGfaUfgUfaAfaAfL96 AD-53119.1 A-108608.1AfcUfuGfgGfaUfCfAfcAfaAfgCfaAfaAfL96 AD-52990.1 A-108334.1UfuGfcUfaUfgUfUfAfgAfcGfaUfgUfaAfL96 AD-52964.1 A-108388.1AfaCfuGfaGfaAfGfAfaCfuAfcAfuAfuAfL96 AD-52973.1 A-108344.1GfaUfgUfaAfaAfAfUfuUfuAfgCfcAfaUfL96 AD-53074.1 A-108546.1AfcUfcCfaUfaGfUfGfaAfgCfaAfuCfuAfL96 AD-53026.1 A-108440.1UfuCfaAfcAfaAfAfAfgUfgAfaAfuAfuUfL96 AD-53062.1 A-108542.1CfuAfgAfgAfaGfAfUfaUfaCfuCfcAfuAfL96 AD-53114.1 A-108622.1CfaAfcCfuAfaAfUfGfgUfaAfaUfaUfaAfL96 AD-53082.1 A-108580.1GfaAfaUfcAfcGfAfAfaCfcAfaCfuAfuAfL96 AD-53035.1 A-108490.1CfcAfcAfgAfaAfUfUfuCfuCfuAfuCfuUfL96 AD-52978.1 A-108330.1AfaAfuCfaAfgAfUfUfuGfcUfaUfgUfuAfL96 AD-53084.1 A-108518.1AfcAfuUfaAfuUfCfAfaCfaUfcGfaAfuAfL96 AD-52972.1 A-108328.1CfcAfgAfgCfcAfAfAfaUfcAfaGfaUfuUfL96 AD-53002.1 A-108432.1CfuAfcUfuCfaAfCfAfaAfaAfgUfgAfaAfL96 AD-53078.1 A-108516.1GfaCfaUfuAfaUfUfCfaAfcAfuCfgAfaUfL96 AD-53072.1 A-108514.1GfgAfcAfuUfaAfUfUfcAfaCfaUfcGfaAfL96 AD-53005.1 A-108480.1GfcAfuAfgUfcAfAfAfuAfaAfaGfaAfaUfL96 AD-53083.1 A-108502.1CfuCfuCfaAfgUfUfUfuUfcAfuGfuCfuAfL96 AD-53102.1 A-108524.1AfuCfgAfaUfaGfAfUfgGfaUfcAfcAfaAfL96 AD-53105.1 A-108572.1AfcAfuUfaUfaUfUfGfaAfuAfuUfcUfuUfL96 AD-53090.1 A-108520.1UfuAfaUfuCfaAfCfAfuCfgAfaUfaGfaUfL96 AD-53010.1 A-108466.1GfaUfaAfuAfgCfAfUfcAfaAfgAfcCfuUfL96 AD-52998.1 A-108368.1UfgAfcAfuAfuUfUfCfaAfaAfaCfuCfaAfL96 AD-52992.1 A-108366.1AfaAfuUfaAfuGfAfCfaUfaUfuUfcAfaAfL96 AD-53068.1 A-108544.1GfaAfgAfuAfuAfCfUfcCfaUfaGfuGfaAfL96 AD-53032.1 A-108442.1AfaUfaUfuUfaGfAfAfgAfgCfaAfcUfaAfL96 AD-52967.1 A-108342.1CfgAfuGfuAfaAfAfAfuUfuUfaGfcCfaAfL96 AD-53096.1 A-108522.1UfuCfaAfcAfuCfGfAfaUfaGfaUfgGfaUfL96 AD-53131.1 A-108612.1GfuGfuGfgAfgAfAfAfaCfaAfcCfuAfaAfL96 AD-52963.1 A-108372.1UfcAfaCfaUfaUfUfUfgAfuCfaGfuCfuUfL96 AD-53089.1 A-108504.1UfcAfgGfuAfgUfCfCfaUfgGfaCfaUfuAfL96 AD-53044.1 A-108446.1UfuUfaGfaAfgAfGfCfaAfcUfaAfcUfaAfL96 AD-52988.1 A-108396.1UfaCfaUfaUfaAfAfCfuAfcAfaGfuCfaAfL96 AD-53067.1 A-108528.1GfgAfuCfaCfaAfAfAfcUfuCfaAfuGfaAfL96 AD-53009.1 A-108450.1AfgAfgCfaAfcUfAfAfcUfaAfcUfuAfaUfL96 AD-53022.1 A-108470.1AfcCfaAfcAfgCfAfUfaGfuCfaAfaUfaAfL96 AD-53016.1 A-108468.1AfaCfcAfaCfaGfCfAfuAfgUfcAfaAfuAfL96 AD-53007.1 A-108418.1GfaAfcUfcAfaCfUfCfaAfaAfcUfuGfaAfL96 AD-53148.1 A-108634.1UfaCfuCfuAfuAfAfAfaUfcAfaCfcAfaAfL96 AD-53040.1 A-108476.1CfaGfcAfuAfgUfCfAfaAfuAfaAfaGfaAfL96 AD-53041.1 A-108492.1GfaAfaUfaAfgAfAfAfuGfuAfaAfaCfaUfL96 AD-53039.1 A-108460.1CfuAfaCfuAfaCfUfUfaAfuUfcAfaAfaUfL96 AD-53139.1 A-108646.1AfuGfaAfcUfgAfGfGfcAfaAfuUfuAfaAfL96 AD-53144.1 A-108648.1UfgAfaCfuGfaGfGfCfaAfaUfuUfaAfaAfL96 AD-53142.1 A-108616.1AfaAfcAfaCfcUfAfAfaUfgGfuAfaAfuAfL96 AD-53108.1 A-108620.1AfcAfaCfcUfaAfAfUfgGfuAfaAfuAfuAfL96 AD-53079.1 A-108532.1AfaCfgUfgGfgAfGfAfaCfuAfcAfaAfuAfL96 AD-53133.1 A-108644.1AfaUfgAfaCfuGfAfGfgCfaAfaUfuUfaAfL96 AD-53104.1 A-108556.1GfuUfgGfaAfgAfCfUfgGfaAfaGfaCfaAfL96 AD-53088.1 A-108582.1UfgGfcAfaUfgUfCfCfcCfaAfuGfcAfaUfL96 AD-53101.1 A-108508.1GfgUfaGfuCfcAfUfGfgAfcAfuUfaAfuUfL96 AD-53000.1 A-108400.1CfaUfaUfaAfaCfUfAfcAfaGfuCfaAfaAfL96 AD-53112.1 A-108590.1AfaUfcCfcGfgAfAfAfaCfaAfaGfaUfuUfL96 AD-53107.1 A-108604.1CfuAfcUfuGfgGfAfUfcAfcAfaAfgCfaAfL96 AD-53121.1 A-108640.1UfgAfaUfgAfaCfUfGfaGfgCfaAfaUfuUfL96 AD-53046.1 A-108478.1AfgCfaUfaGfuCfAfAfaUfaAfaAfgAfaAfL96 AD-53038.1 A-108444.1AfuUfuAfgAfaGfAfGfcAfaCfuAfaCfuAfL96 AD-53140.1 A-108662.1AfgGfcAfaAfuUfUfAfaAfaGfgCfaAfuAfL96 AD-52987.1 A-108380.1CfaUfaUfuUfgAfUfCfaGfuCfuUfuUfuAfL96 AD-53130.1 A-108596.1AfaAfaCfaAfaGfAfUfuUfgGfuGfuUfuUfL96 AD-53106.1 A-108588.1CfaAfuCfcCfgGfAfAfaAfcAfaAfgAfuUfL96 AD-53081.1 A-108564.1CfaAfcAfaAfcAfUfUfaUfaUfuGfaAfuAfL96 AD-53118.1 A-108592.1GfgAfaAfaCfaAfAfGfaUfuUfgGfuGfuUfL96 AD-53136.1 A-108598.1AfcAfaAfgAfuUfUfGfgUfgUfuUfuCfuAfL96 AD-53127.1 A-108642.1GfaAfuGfaAfcUfGfAfgGfcAfaAfuUfuAfL96 AD-53066.1 A-108512.1CfcAfuGfgAfcAfUfUfaAfuUfcAfaCfaUfL96 AD-53013.1 A-108420.1AfaCfuCfaAfcUfCfAfaAfaCfuUfgAfaAfL96 AD-52991.1 A-108350.1CfaGfuUfgGfgAfCfAfuGfgUfcUfuAfaAfL96 AD-53099.1 A-108570.1AfaCfaUfuAfuAfUfUfgAfaUfaUfuCfuUfL96 AD-52958.1 A-108386.1AfcCfaGfuGfaAfAfUfcAfaAfgAfaGfaAfL96 AD-53097.1 A-108538.1GfuUfgGfgCfcUfAfGfaGfaAfgAfuAfuAfL96 AD-52966.1 A-108326.1CfuCfcAfgAfgCfCfAfaAfaUfcAfaGfaUfL96 AD-53145.1 A-108664.1GfgCfaAfaUfuUfAfAfaAfgGfcAfaUfaAfL96 AD-53113.1 A-108606.1UfaCfuUfgGfgAfUfCfaCfaAfaGfcAfaAfL96 AD-52993.1 A-108382.1GfaUfcAfgUfcUfUfUfuUfaUfgAfuCfuAfL96 AD-53031.1 A-108426.1GfaAfaGfcCfuCfCfUfaGfaAfgAfaAfaAfL96 AD-53017.1 A-108484.1AfgUfcAfaAfuAfAfAfaGfaAfaUfaGfaAfL96 AD-53143.1 A-108632.1AfuAfcUfcUfaUfAfAfaAfuCfaAfcCfaAfL96 AD-53149.1 A-108650.1GfaAfcUfgAfgGfCfAfaAfuUfuAfaAfaAfL96 AD-53059.1 A-108494.1AfgAfcCfcAfgCfAfAfcUfcUfcAfaGfuUfL96 AD-53006.1 A-108402.1AfuAfuAfaAfcUfAfCfaAfgUfcAfaAfaAfL96 AD-53025.1 A-108424.1UfgAfaAfgCfcUfCfCfuAfgAfaGfaAfaAfL96 AD-53085.1 A-108534.1GfgGfaGfaAfcUfAfCfaAfaUfaUfgGfuUfL96 AD-52984.1 A-108332.1AfgAfuUfuGfcUfAfUfgUfuAfgAfcGfaUfL96 AD-53023.1 A-108486.1GfaAfcCfcAfcAfGfAfaAfuUfuCfuCfuAfL96 AD-53014.1 A-108436.1AfcUfuCfaAfcAfAfAfaAfgUfgAfaAfuAfL96 AD-53060.1 A-108510.1AfgUfcCfaUfgGfAfCfaUfuAfaUfuCfaAfL96 AD-53110.1 A-108652.1AfaCfuGfaGfgCfAfAfaUfuUfaAfaAfgAfL96 AD-52980.1 A-108362.1GfgGfcCfaAfaUfUfAfaUfgAfcAfuAfuUfL96 AD-53109.1 A-108636.1AfuCfcAfuCfcAfAfCfaGfaUfuCfaGfaAfL96 AD-53141.1 A-108600.1AfaGfaUfuUfgGfUfGfuUfuUfcUfaCfuUfL96 AD-53126.1 A-108626.1GfuCfuCfaAfaAfUfGfgAfaGfgUfuAfuAfL96 AD-53116.1 A-108654.1AfcUfgAfgGfcAfAfAfuUfuAfaAfaGfgAfL96 AD-52997.1 A-108352.1GfgGfaCfaUfgGfUfCfuUfaAfaGfaCfuUfL96 AD-53120.1 A-108624.1AfuGfgUfaAfaUfAfUfaAfcAfaAfcCfaAfL96 AD-53070.1 A-108576.1GfgGfaAfaUfcAfCfGfaAfaCfcAfaCfuAfL96 AD-53028.1 A-108472.1CfcAfaCfaGfcAfUfAfgUfcAfaAfuAfaAfL96 AD-53146.1 A-108602.1UfuUfuCfuAfcUfUfGfgGfaUfcAfcAfaAfL96 AD-52982.1 A-108394.1AfgAfaCfuAfcAfUfAfuAfaAfcUfaCfaAfL96 AD-53111.1 A-108668.1AfgAfgUfaUfgUfGfUfaAfaAfaUfcUfgUfL96 AD-53045.1 A-108462.1AfaAfaCfaAfgAfUfAfaUfaGfcAfuCfaAfL96 AD-53123.1 A-108672.1AfgUfaUfgUfgUfAfAfaAfaUfcUfgUfaAfL96 AD-53018.1 A-108406.1AfgUfcAfaAfaAfUfGfaAfgAfgGfuAfaAfL96 AD-52956.1 A-108354.1GfgAfcAfuGfgUfCfUfuAfaAfgAfcUfuUfL96 AD-53134.1 A-108660.1GfaGfgCfaAfaUfUfUfaAfaAfgGfcAfaUfL96 AD-52968.1 A-108358.1GfuCfuUfaAfaGfAfCfuUfuGfuCfcAfuAfL96 AD-53122.1 A-108656.1CfuGfaGfgCfaAfAfUfuUfaAfaAfgGfcAfL96 AD-53100.1 A-108586.1GfcAfaUfcCfcGfGfAfaAfaCfaAfaGfaUfL96 AD-53128.1 A-108658.1UfgAfgGfcAfaAfUfUfuAfaAfaGfgCfaAfL96 AD-53043.1 A-108430.1UfcUfaCfuUfcAfAfCfaAfaAfaGfuGfaAfL96 AD-53135.1 A-108676.1UfaUfgUfgUfaAfAfAfaUfcUfgUfaAfuAfL96 AD-53094.1 A-108584.1AfaUfgCfaAfuCfCfCfgGfaAfaAfcAfaAfL96 AD-53019.1 A-108422.1CfuUfgAfaAfgCfCfUfcCfuAfgAfaGfaAfL96 AD-53129.1 A-108674.1GfuAfuGfuGfuAfAfAfaAfuCfuGfuAfaUfL96 AD-53150.1 A-108666.1CfaGfaGfuAfuGfUfGfuAfaAfaAfuCfuUfL96 AD-53117.1 A-108670.1GfaGfuAfuGfuGfUfAfaAfaAfuCfuGfuAfL96 AD-52985.1 A-108348.1UfcAfgUfuGfgGfAfCfaUfgGfuCfuUfaAfL96 AD-52962.1 A-108356.1GfgUfcUfuAfaAfGfAfcUfuUfgUfcCfaUfL96 AD-52974.1 A-108360.1UfcUfuAfaAfgAfCfUfuUfgUfcCfaUfaAfL96 AD-52979.1 A-108346.1UfuCfaGfuUfgGfGfAfcAfuGfgUfcUfuAfL96 Antisense Sequence Antisense(SEQ ID NOS 819-1003, respectively, in Duplex ID OligoNameorder of appearance) AD-53063.1 A-108559.1aAfuAfuAfaUfgUfuugUfuGfuCfuUfusCfsc AD-52965.1 A-108311.1aAfaAfaGfaAfgGfagcUfuAfaUfuGfusGfsa AD-53030.1 A-108411.1uUfgAfgUfuGfaGfuucAfaGfuGfaCfasUfsa AD-52953.1 A-108307.1aAfaGfaAfgGfaGfcuuAfaUfuGfuGfasAfsc AD-53001.1 A-108417.1aAfgUfuUfuGfaGfuugAfgUfuCfaAfgsUfsg AD-53080.1 A-108549.1uUfaGfaUfuGfcUfucaCfuAfuGfgAfgsUfsa AD-52971.1 A-108313.1uAfaAfaAfgAfaGfgagCfuUfaAfuUfgsUfsg AD-53071.1 A-108499.1aAfaAfcUfuGfaGfaguUfgCfuGfgGfusCfsu AD-53024.1 A-108409.1uUfgAfgUfuCfaAfgugAfcAfuAfuUfcsUfsu AD-52977.1 A-108315.1aUfaAfaAfaGfaAfggaGfcUfuAfaUfusGfsu AD-53064.1 A-108575.1aAfaAfgAfaUfaUfucaAfuAfuAfaUfgsUfsu AD-53033.1 A-108459.1uUfuUfgAfaUfuAfaguUfaGfuUfaGfusUfsg AD-52954.1 A-108323.1aAfaUfaAfcUfaGfaggAfaCfaAfuAfasAfsa AD-53098.1 A-108555.1uAfaUfuAfgAfuUfgcuUfcAfcUfaUfgsGfsa AD-53092.1 A-108553.1aAfuUfaGfaUfuGfcuuCfaCfuAfuGfgsAfsg AD-53073.1 A-108531.1uUfuCfaUfuGfaAfguuUfuGfuGfaUfcsCfsa AD-53132.1 A-108629.1uUfaUfaGfaGfuAfuaaCfcUfuCfcAfusUfsu AD-53086.1 A-108551.1aUfuAfgAfuUfgCfuucAfcUfaUfgGfasGfsu AD-52961.1 A-108341.1uUfuUfuAfcAfuCfgucUfaAfcAfuAfgsCfsa AD-52983.1 A-108317.1aAfuAfaAfaAfgAfaggAfgCfuUfaAfusUfsg AD-53027.1 A-108457.1uUfuGfaAfuUfaAfguuAfgUfuAfgUfusGfsc AD-52986.1 A-108365.1aAfaUfaUfgUfcAfuuaAfuUfuGfgCfcsCfsu AD-52989.1 A-108319.1aUfaAfcUfaGfaGfgaaCfaAfuAfaAfasAfsg AD-52981.1 A-108379.1aAfaAfaGfaCfuGfaucAfaAfuAfuGfusUfsg AD-53077.1 A-108501.1aAfaAfaCfuUfgAfgagUfuGfcUfgGfgsUfsc AD-53095.1 A-108507.1uUfaAfuGfuCfcAfuggAfcUfaCfcUfgsAfsu AD-52970.1 A-108391.1uUfaUfaUfgUfaGfuucUfuCfuCfaGfusUfsc AD-53015.1 A-108453.1aAfuUfaAfgUfuAfguuAfgUfuGfcUfcsUfsu AD-53147.1 A-108619.1aUfaUfuUfaCfcAfuuuAfgGfuUfgUfusUfsu AD-53103.1 A-108541.1aUfgGfaGfuAfuAfucuUfcUfcUfaGfgsCfsc AD-52969.1 A-108375.1aAfaGfaCfuGfaUfcaaAfuAfuGfuUfgsAfsg AD-53075.1 A-108563.1aUfuCfaAfuAfuAfaugUfuUfgUfuGfusCfsu AD-52994.1 A-108399.1uUfuGfaCfuUfgUfaguUfuAfuAfuGfusAfsg AD-52960.1 A-108325.1aAfuUfcUfgGfaGfgaaAfuAfaCfuAfgsAfsg AD-53003.1 A-108449.1uUfaAfgUfuAfgUfuagUfuGfcUfcUfusCfsu AD-52995.1 A-108321.1aAfuAfaCfuAfgAfggaAfcAfaUfaAfasAfsa AD-53037.1 A-108429.1uAfgAfaUfuUfuUfucuUfcUfaGfgAfgsGfsc AD-53087.1 A-108567.1aUfaUfuCfaAfuAfuaaUfgUfuUfgUfusGfsu AD-53076.1 A-108579.1aUfaGfuUfgGfuUfucgUfgAfuUfuCfcsCfsa AD-52975.1 A-108377.1aAfaAfgAfcUfgAfucaAfaUfaUfgUfusGfsa AD-53138.1 A-108631.1uUfuAfuAfgAfgUfauaAfcCfuUfcCfasUfsu AD-53091.1 A-108537.1aAfaCfcAfuAfuUfuguAfgUfuCfuCfcsCfsa AD-53124.1 A-108595.1aAfaCfaCfcAfaAfucuUfuGfuUfuUfcsCfsg AD-53125.1 A-108611.1uUfaGfgUfuGfuUfuucUfcCfaCfaCfusCfsa AD-53036.1 A-108413.1uUfuGfaGfuUfgAfguuCfaAfgUfgAfcsAfsu AD-53061.1 A-108527.1aUfuGfaAfgUfuUfuguGfaUfcCfaUfcsUfsa AD-53093.1 A-108569.1aAfuAfuUfcAfaUfauaAfuGfuUfuGfusUfsg AD-53137.1 A-108615.1aUfuUfaGfgUfuGfuuuUfcUfcCfaCfasCfsu AD-52999.1 A-108385.1aUfaGfaUfcAfuAfaaaAfgAfcUfgAfusCfsa AD-53069.1 A-108561.1uUfcAfaUfaUfaAfuguUfuGfuUfgUfcsUfsu AD-53034.1 A-108475.1uUfuUfaUfuUfgAfcuaUfgCfuGfuUfgsGfsu AD-52976.1 A-108393.1uUfuAfuAfuGfuAfguuCfuUfcUfcAfgsUfsu AD-52996.1 A-108337.1uUfuAfcAfuCfgUfcuaAfcAfuAfgCfasAfsa AD-53029.1 A-108489.1aUfaGfaGfaAfaUfuucUfgUfgGfgUfusCfsu AD-53020.1 A-108439.1aUfaUfuUfcAfcUfuuuUfgUfuGfaAfgsUfsa AD-53042.1 A-108415.1uUfuUfgAfgUfuGfaguUfcAfaGfuGfasCfsa AD-53011.1 A-108483.1uAfuUfuCfuUfuUfauuUfgAfcUfaUfgsCfsu AD-52957.1 A-108371.1aUfcAfaAfuAfuGfuugAfgUfuUfuUfgsAfsa AD-53008.1 A-108435.1aUfuUfcAfcUfuUfuugUfuGfaAfgUfasGfsa AD-53065.1 A-108497.1aAfaCfuUfgAfgAfguuGfcUfgGfgUfcsUfsg AD-53115.1 A-108639.1aAfuUfuGfcCfuCfaguUfcAfuUfcAfasAfsg AD-53012.1 A-108405.1aUfuUfuUfgAfcUfuguAfgUfuUfaUfasUfsg AD-53004.1 A-108465.1uUfuGfaUfgCfuAfuuaUfcUfuGfuUfusUfsu AD-53021.1 A-108455.1uUfgAfaUfuAfaGfuuaGfuUfaGfuUfgsCfsu AD-52955.1 A-108339.1uUfuUfaCfaUfcGfucuAfaCfaUfaGfcsAfsa AD-53119.1 A-108609.1uUfuUfgCfuUfuGfugaUfcCfcAfaGfusAfsg AD-52990.1 A-108335.1uUfaCfaUfcGfuCfuaaCfaUfaGfcAfasAfsu AD-52964.1 A-108389.1uAfuAfuGfuAfgUfucuUfcUfcAfgUfusCfsc AD-52973.1 A-108345.1aUfuGfgCfuAfaAfauuUfuUfaCfaUfcsGfsu AD-53074.1 A-108547.1uAfgAfuUfgCfuUfcacUfaUfgGfaGfusAfsu AD-53026.1 A-108441.1aAfuAfuUfuCfaCfuuuUfuGfuUfgAfasGfsu AD-53062.1 A-108543.1uAfuGfgAfgUfaUfaucUfuCfuCfuAfgsGfsc AD-53114.1 A-108623.1uUfaUfaUfuUfaCfcauUfuAfgGfuUfgsUfsu AD-53082.1 A-108581.1uAfuAfgUfuGfgUfuucGfuGfaUfuUfcsCfsc AD-53035.1 A-108491.1aAfgAfuAfgAfgAfaauUfuCfuGfuGfgsGfsu AD-52978.1 A-108331.1uAfaCfaUfaGfcAfaauCfuUfgAfuUfusUfsg AD-53084.1 A-108519.1uAfuUfcGfaUfgUfugaAfuUfaAfuGfusCfsc AD-52972.1 A-108329.1aAfaUfcUfuGfaUfuuuGfgCfuCfuGfgsAfsg AD-53002.1 A-108433.1uUfuCfaCfuUfuUfuguUfgAfaGfuAfgsAfsa AD-53078.1 A-108517.1aUfuCfgAfuGfuUfgaaUfuAfaUfgUfcsCfsa AD-53072.1 A-108515.1uUfcGfaUfgUfuGfaauUfaAfuGfuCfcsAfsu AD-53005.1 A-108481.1aUfuUfcUfuUfuAfuuuGfaCfuAfuGfcsUfsg AD-53083.1 A-108503.1uAfgAfcAfuGfaAfaaaCfuUfgAfgAfgsUfsu AD-53102.1 A-108525.1uUfuGfuGfaUfcCfaucUfaUfuCfgAfusGfsu AD-53105.1 A-108573.1aAfaGfaAfuAfuUfcaaUfaUfaAfuGfusUfsu AD-53090.1 A-108521.1aUfcUfaUfuCfgAfuguUfgAfaUfuAfasUfsg AD-53010.1 A-108467.1aAfgGfuCfuUfuGfaugCfuAfuUfaUfcsUfsu AD-52998.1 A-108369.1uUfgAfgUfuUfuUfgaaAfuAfuGfuCfasUfsu AD-52992.1 A-108367.1uUfuGfaAfaUfaUfgucAfuUfaAfuUfusGfsg AD-53068.1 A-108545.1uUfcAfcUfaUfgGfaguAfuAfuCfuUfcsUfsc AD-53032.1 A-108443.1uUfaGfuUfgCfuCfuucUfaAfaUfaUfusUfsc AD-52967.1 A-108343.1uUfgGfcUfaAfaAfuuuUfuAfcAfuCfgsUfsc AD-53096.1 A-108523.1aUfcCfaUfcUfaUfucgAfuGfuUfgAfasUfsu AD-53131.1 A-108613.1uUfuAfgGfuUfgUfuuuCfuCfcAfcAfcsUfsc AD-52963.1 A-108373.1aAfgAfcUfgAfuCfaaaUfaUfgUfuGfasGfsu AD-53089.1 A-108505.1uAfaUfgUfcCfaUfggaCfuAfcCfuGfasUfsa AD-53044.1 A-108447.1uUfaGfuUfaGfuUfgcuCfuUfcUfaAfasUfsa AD-52988.1 A-108397.1uUfgAfcUfuGfuAfguuUfaUfaUfgUfasGfsu AD-53067.1 A-108529.1uUfcAfuUfgAfaGfuuuUfgUfgAfuCfcsAfsu AD-53009.1 A-108451.1aUfuAfaGfuUfaGfuuaGfuUfgCfuCfusUfsc AD-53022.1 A-108471.1uUfaUfuUfgAfcUfaugCfuGfuUfgGfusUfsu AD-53016.1 A-108469.1uAfuUfuGfaCfuAfugcUfgUfuGfgUfusUfsa AD-53007.1 A-108419.1uUfcAfaGfuUfuUfgagUfuGfaGfuUfcsAfsa AD-53148.1 A-108635.1uUfuGfgUfuGfaUfuuuAfuAfgAfgUfasUfsa AD-53040.1 A-108477.1uUfcUfuUfuAfuUfugaCfuAfuGfcUfgsUfsu AD-53041.1 A-108493.1aUfgUfuUfuAfcAfuuuCfuUfaUfuUfcsAfsu AD-53039.1 A-108461.1aUfuUfuGfaAfuUfaagUfuAfgUfuAfgsUfsu AD-53139.1 A-108647.1uUfuAfaAfuUfuGfccuCfaGfuUfcAfusUfsc AD-53144.1 A-108649.1uUfuUfaAfaUfuUfgccUfcAfgUfuCfasUfsu AD-53142.1 A-108617.1uAfuUfuAfcCfaUfuuaGfgUfuGfuUfusUfsc AD-53108.1 A-108621.1uAfuAfuUfuAfcCfauuUfaGfgUfuGfusUfsu AD-53079.1 A-108533.1uAfuUfuGfuAfgUfucuCfcCfaCfgUfusUfsc AD-53133.1 A-108645.1uUfaAfaUfuUfgCfcucAfgUfuCfaUfusCfsa AD-53104.1 A-108557.1uUfgUfcUfuUfcCfaguCfuUfcCfaAfcsUfsc AD-53088.1 A-108583.1aUfuGfcAfuUfgGfggaCfaUfuGfcCfasGfsu AD-53101.1 A-108509.1aAfuUfaAfuGfuCfcauGfgAfcUfaCfcsUfsg AD-53000.1 A-108401.1uUfuUfgAfcUfuGfuagUfuUfaUfaUfgsUfsa AD-53112.1 A-108591.1aAfaUfcUfuUfgUfuuuCfcGfgGfaUfusGfsc AD-53107.1 A-108605.1uUfgCfuUfuGfuGfaucCfcAfaGfuAfgsAfsa AD-53121.1 A-108641.1aAfaUfuUfgCfcUfcagUfuCfaUfuCfasAfsa AD-53046.1 A-108479.1uUfuCfuUfuUfaUfuugAfcUfaUfgCfusGfsu AD-53038.1 A-108445.1uAfgUfuAfgUfuGfcucUfuCfuAfaAfusAfsu AD-53140.1 A-108663.1uAfuUfgCfcUfuUfuaaAfuUfuGfcCfusCfsa AD-52987.1 A-108381.1uAfaAfaAfgAfcUfgauCfaAfaUfaUfgsUfsu AD-53130.1 A-108597.1aAfaAfcAfcCfaAfaucUfuUfgUfuUfusCfsc AD-53106.1 A-108589.1aAfuCfuUfuGfuUfuucCfgGfgAfuUfgsCfsa AD-53081.1 A-108565.1uAfuUfcAfaUfaUfaauGfuUfuGfuUfgsUfsc AD-53118.1 A-108593.1aAfcAfcCfaAfaUfcuuUfgUfuUfuCfcsGfsg AD-53136.1 A-108599.1uAfgAfaAfaCfaCfcaaAfuCfuUfuGfusUfsu AD-53127.1 A-108643.1uAfaAfuUfuGfcCfucaGfuUfcAfuUfcsAfsa AD-53066.1 A-108513.1aUfgUfuGfaAfuUfaauGfuCfcAfuGfgsAfsc AD-53013.1 A-108421.1uUfuCfaAfgUfuUfugaGfuUfgAfgUfusCfsa AD-52991.1 A-108351.1uUfuAfaGfaCfcAfuguCfcCfaAfcUfgsAfsa AD-53099.1 A-108571.1aAfgAfaUfaUfuCfaauAfuAfaUfgUfusUfsg AD-52958.1 A-108387.1uUfcUfuCfuUfuGfauuUfcAfcUfgGfusUfsu AD-53097.1 A-108539.1uAfuAfuCfuUfcUfcuaGfgCfcCfaAfcsCfsa AD-52966.1 A-108327.1aUfcUfuGfaUfuUfuggCfuCfuGfgAfgsAfsu AD-53145.1 A-108665.1uUfaUfuGfcCfuUfuuaAfaUfuUfgCfcsUfsc AD-53113.1 A-108607.1uUfuGfcUfuUfgUfgauCfcCfaAfgUfasGfsa AD-52993.1 A-108383.1uAfgAfuCfaUfaAfaaaGfaCfuGfaUfcsAfsa AD-53031.1 A-108427.1uUfuUfuCfuUfcUfaggAfgGfcUfuUfcsAfsa AD-53017.1 A-108485.1uUfcUfaUfuUfcUfuuuAfuUfuGfaCfusAfsu AD-53143.1 A-108633.1uUfgGfuUfgAfuUfuuaUfaGfaGfuAfusAfsa AD-53149.1 A-108651.1uUfuUfuAfaAfuUfugcCfuCfaGfuUfcsAfsu AD-53059.1 A-108495.1aAfcUfuGfaGfaGfuugCfuGfgGfuCfusGfsa AD-53006.1 A-108403.1uUfuUfuGfaCfuUfguaGfuUfuAfuAfusGfsu AD-53025.1 A-108425.1uUfuUfcUfuCfuAfggaGfgCfuUfuCfasAfsg AD-53085.1 A-108535.1aAfcCfaUfaUfuUfguaGfuUfcUfcCfcsAfsc AD-52984.1 A-108333.1aUfcGfuCfuAfaCfauaGfcAfaAfuCfusUfsg AD-53023.1 A-108487.1uAfgAfgAfaAfuUfucuGfuGfgGfuUfcsUfsu AD-53014.1 A-108437.1uAfuUfuCfaCfuUfuuuGfuUfgAfaGfusAfsg AD-53060.1 A-108511.1uUfgAfaUfuAfaUfgucCfaUfgGfaCfusAfsc AD-53110.1 A-108653.1uCfuUfuUfaAfaUfuugCfcUfcAfgUfusCfsa AD-52980.1 A-108363.1aAfuAfuGfuCfaUfuaaUfuUfgGfcCfcsUfsu AD-53109.1 A-108637.1uUfcUfgAfaUfcUfguuGfgAfuGfgAfusCfsa AD-53141.1 A-108601.1aAfgUfaGfaAfaAfcacCfaAfaUfcUfusUfsg AD-53126.1 A-108627.1uAfuAfaCfcUfuCfcauUfuUfgAfgAfcsUfsu AD-53116.1 A-108655.1uCfcUfuUfuAfaAfuuuGfcCfuCfaGfusUfsc AD-52997.1 A-108353.1aAfgUfcUfuUfaAfgacCfaUfgUfcCfcsAfsa AD-53120.1 A-108625.1uUfgGfuUfuGfuUfauaUfuUfaCfcAfusUfsu AD-53070.1 A-108577.1uAfgUfuGfgUfuUfcguGfaUfuUfcCfcsAfsa AD-53028.1 A-108473.1uUfuAfuUfuGfaCfuauGfcUfgUfuGfgsUfsu AD-53146.1 A-108603.1uUfuGfuGfaUfcCfcaaGfuAfgAfaAfasCfsa AD-52982.1 A-108395.1uUfgUfaGfuUfuAfuauGfuAfgUfuCfusUfsc AD-53111.1 A-108669.1aCfaGfaUfuUfuUfacaCfaUfaCfuCfusGfsu AD-53045.1 A-108463.1uUfgAfuGfcUfaUfuauCfuUfgUfuUfusUfsc AD-53123.1 A-108673.1uUfaCfaGfaUfuUfuuaCfaCfaUfaCfusCfsu AD-53018.1 A-108407.1uUfuAfcCfuCfuUfcauUfuUfuGfaCfusUfsg AD-52956.1 A-108355.1aAfaGfuCfuUfuAfagaCfcAfuGfuCfcsCfsa AD-53134.1 A-108661.1aUfuGfcCfuUfuUfaaaUfuUfgCfcUfcsAfsg AD-52968.1 A-108359.1uAfuGfgAfcAfaAfgucUfuUfaAfgAfcsCfsa AD-53122.1 A-108657.1uGfcCfuUfuUfaAfauuUfgCfcUfcAfgsUfsu AD-53100.1 A-108587.1aUfcUfuUfgUfuUfuccGfgGfaUfuGfcsAfsu AD-53128.1 A-108659.1uUfgCfcUfuUfuAfaauUfuGfcCfuCfasGfsu AD-53043.1 A-108431.1uUfcAfcUfuUfuUfguuGfaAfgUfaGfasAfsu AD-53135.1 A-108677.1uAfuUfaCfaGfaUfuuuUfaCfaCfaUfasCfsu AD-53094.1 A-108585.1uUfuGfuUfuUfcCfgggAfuUfgCfaUfusGfsg AD-53019.1 A-108423.1uUfcUfuCfuAfgGfaggCfuUfuCfaAfgsUfsu AD-53129.1 A-108675.1aUfuAfcAfgAfuUfuuuAfcAfcAfuAfcsUfsc AD-53150.1 A-108667.1aAfgAfuUfuUfuAfcacAfuAfcUfcUfgsUfsg AD-53117.1 A-108671.1uAfcAfgAfuUfuUfuacAfcAfuAfcUfcsUfsg AD-52985.1 A-108349.1uUfaAfgAfcCfaUfgucCfcAfaCfuGfasAfsg AD-52962.1 A-108357.1aUfgGfaCfaAfaGfucuUfuAfaGfaCfcsAfsu AD-52974.1 A-108361.1uUfaUfgGfaCfaAfaguCfuUfuAfaGfasCfsc AD-52979.1 A-108347.1uAfaGfaCfcAfuGfuccCfaAfcUfgAfasGfsg Lowercase nucleotides (a, u, g, c)are 2′-O-methyl nucleotides; Nf (e.g., Af) is a 2′-fluoro nucleotide; sis a phosphothiorate linkage; L96 indicates a GalNAc ligand.

TABLE 9Unmodified Sense and antisense strand sequences of ANGPTL3 dsRNAs without GalNal conjugationThese sequences are the same as the sequences listed inTable 7 except that they do not contain GalNal conjugation.Sense Sequence Antisense Sequence (SEQ ID NOS 1004-1184,(SEQ ID NOS 1185-1365, Sense respectively, in order of Antisenserespectively, in order of Position in Duplex Name OligoName appearance)OligoName appearance) NM_014495.2 AD-52637.1 A-108817.1UCACAAUUAAGCUCCUUCUUU A-108307.2 AAAGAAGGAGCUUAAUUGUGAAC 54-76AD-52638.1 A-108825.1 UUAUUGUUCCUCUAGUUAUUU A-108323.2AAAUAACUAGAGGAACAAUAAAA 75-97 AD-52639.1 A-108833.1GCUAUGUUAGACGAUGUAAAA A-108339.2 UUUUACAUCGUCUAACAUAGCAA 161-183AD-52640.1 A-108841.1 GGACAUGGUCUUAAAGACUUU A-108355.2AAAGUCUUUAAGACCAUGUCCCA 209-231 AD-52641.1 A-108849.1CAAAAACUCAACAUAUUUGAU A-108371.2 AUCAAAUAUGUUGAGUUUUUGAA 266-288AD-52642.1 A-108857.1 ACCAGUGAAAUCAAAGAAGAA A-108387.2UUCUUCUUUGAUUUCACUGGUUU 314-336 AD-52643.1 A-108818.1CACAAUUAAGCUCCUUCUUUU A-108309.2 AAAAGAAGGAGCUUAAUUGUGAA 55-77AD-52645.1 A-108834.1 CUAUGUUAGACGAUGUAAAAA A-108341.2UUUUUACAUCGUCUAACAUAGCA 162-184 AD-52647.1 A-108850.1UCAACAUAUUUGAUCAGUCUU A-108373.2 AAGACUGAUCAAAUAUGUUGAGU 273-295AD-52648.1 A-108858.1 AACUGAGAAGAACUACAUAUA A-108389.2UAUAUGUAGUUCUUCUCAGUUCC 342-364 AD-52649.1 A-108819.1ACAAUUAAGCUCCUUCUUUUU A-108311.2 AAAAAGAAGGAGCUUAAUUGUGA 56-78AD-52650.1 A-108827.1 CUCCAGAGCCAAAAUCAAGAU A-108327.2AUCUUGAUUUUGGCUCUGGAGAU 138-160 AD-52651.1 A-108835.1CGAUGUAAAAAUUUUAGCCAA A-108343.2 UUGGCUAAAAUUUUUACAUCGUC 172-194AD-52652.1 A-108843.1 GUCUUAAAGACUUUGUCCAUA A-108359.2UAUGGACAAAGUCUUUAAGACCA 216-238 AD-52653.1 A-108851.1CAACAUAUUUGAUCAGUCUUU A-108375.2 AAAGACUGAUCAAAUAUGUUGAG 274-296AD-52654.1 A-108859.1 ACUGAGAAGAACUACAUAUAA A-108391.2UUAUAUGUAGUUCUUCUCAGUUC 343-365 AD-52656.1 A-108828.1CCAGAGCCAAAAUCAAGAUUU A-108329.2 AAAUCUUGAUUUUGGCUCUGGAG 140-162AD-52657.1 A-108836.1 GAUGUAAAAAUUUUAGCCAAU A-108345.2AUUGGCUAAAAUUUUUACAUCGU 173-195 AD-52658.1 A-108844.1UCUUAAAGACUUUGUCCAUAA A-108361.2 UUAUGGACAAAGUCUUUAAGACC 217-239AD-52659.1 A-108852.1 AACAUAUUUGAUCAGUCUUUU A-108377.2AAAAGACUGAUCAAAUAUGUUGA 275-297 AD-52660.1 A-108860.1CUGAGAAGAACUACAUAUAAA A-108393.2 UUUAUAUGUAGUUCUUCUCAGUU 344-366AD-52661.1 A-108821.1 AAUUAAGCUCCUUCUUUUUAU A-108315.2AUAAAAAGAAGGAGCUUAAUUGU 58-80 AD-52662.1 A-108829.1AAAUCAAGAUUUGCUAUGUUA A-108331.2 UAACAUAGCAAAUCUUGAUUUUG 149-171AD-52663.1 A-108837.1 UUCAGUUGGGACAUGGUCUUA A-108347.2UAAGACCAUGUCCCAACUGAAGG 201-223 AD-52664.1 A-108845.1GGGCCAAAUUAAUGACAUAUU A-108363.2 AAUAUGUCAUUAAUUUGGCCCUU 244-266AD-52665.1 A-108853.1 ACAUAUUUGAUCAGUCUUUUU A-108379.2AAAAAGACUGAUCAAAUAUGUUG 276-298 AD-52666.1 A-108861.1AGAACUACAUAUAAACUACAA A-108395.2 UUGUAGUUUAUAUGUAGUUCUUC 350-372AD-52667.1 A-108822.1 AUUAAGCUCCUUCUUUUUAUU A-108317.2AAUAAAAAGAAGGAGCUUAAUUG 59-81 AD-52668.1 A-108830.1AGAUUUGCUAUGUUAGACGAU A-108333.2 AUCGUCUAACAUAGCAAAUCUUG 155-177AD-52669.1 A-108838.1 UCAGUUGGGACAUGGUCUUAA A-108349.2UUAAGACCAUGUCCCAACUGAAG 202-224 AD-52670.1 A-108846.1GGCCAAAUUAAUGACAUAUUU A-108365.2 AAAUAUGUCAUUAAUUUGGCCCU 245-267AD-52671.1 A-108854.1 CAUAUUUGAUCAGUCUUUUUA A-108381.2UAAAAAGACUGAUCAAAUAUGUU 277-299 AD-52672.1 A-108862.1UACAUAUAAACUACAAGUCAA A-108397.2 UUGACUUGUAGUUUAUAUGUAGU 355-377AD-52673.1 A-108823.1 UUUUAUUGUUCCUCUAGUUAU A-108319.2AUAACUAGAGGAACAAUAAAAAG 73-95 AD-52674.1 A-108831.1UUGCUAUGUUAGACGAUGUAA A-108335.2 UUACAUCGUCUAACAUAGCAAAU 159-181AD-52675.1 A-108839.1 CAGUUGGGACAUGGUCUUAAA A-108351.2UUUAAGACCAUGUCCCAACUGAA 203-225 AD-52676.1 A-108847.1AAAUUAAUGACAUAUUUCAAA A-108367.2 UUUGAAAUAUGUCAUUAAUUUGG 249-271AD-52677.1 A-108855.1 GAUCAGUCUUUUUAUGAUCUA A-108383.2UAGAUCAUAAAAAGACUGAUCAA 284-306 AD-52678.1 A-108863.1ACAUAUAAACUACAAGUCAAA A-108399.2 UUUGACUUGUAGUUUAUAUGUAG 356-378AD-52679.1 A-108824.1 UUUAUUGUUCCUCUAGUUAUU A-108321.2AAUAACUAGAGGAACAAUAAAAA 74-96 AD-52680.1 A-108832.1UGCUAUGUUAGACGAUGUAAA A-108337.2 UUUACAUCGUCUAACAUAGCAAA 160-182AD-52681.1 A-108840.1 GGGACAUGGUCUUAAAGACUU A-108353.2AAGUCUUUAAGACCAUGUCCCAA 208-230 AD-52682.1 A-108848.1UGACAUAUUUCAAAAACUCAA A-108369.2 UUGAGUUUUUGAAAUAUGUCAUU 256-278AD-52683.1 A-108856.1 AUCAGUCUUUUUAUGAUCUAU A-108385.2AUAGAUCAUAAAAAGACUGAUCA 285-307 AD-52684.1 A-108864.1CAUAUAAACUACAAGUCAAAA A-108401.2 UUUUGACUUGUAGUUUAUAUGUA 357-379AD-52685.1 A-108872.1 CUUGAACUCAACUCAAAACUU A-108417.2AAGUUUUGAGUUGAGUUCAAGUG 401-423 AD-52686.1 A-108880.1CUACUUCAACAAAAAGUGAAA A-108433.2 UUUCACUUUUUGUUGAAGUAGAA 446-468AD-52687.1 A-108888.1 AAGAGCAACUAACUAACUUAA A-108449.2UUAAGUUAGUUAGUUGCUCUUCU 474-496 AD-52688.1 A-108896.1AAACAAGAUAAUAGCAUCAAA A-108465.2 UUUGAUGCUAUUAUCUUGUUUUU 557-579AD-52689.1 A-108904.1 GCAUAGUCAAAUAAAAGAAAU A-108481.2AUUUCUUUUAUUUGACUAUGCUG 625-647 AD-52690.1 A-108865.1AUAUAAACUACAAGUCAAAAA A-108403.2 UUUUUGACUUGUAGUUUAUAUGU 358-380AD-52691.1 A-108873.1 GAACUCAACUCAAAACUUGAA A-108419.2UUCAAGUUUUGAGUUGAGUUCAA 404-426 AD-52692.1 A-108881.1UACUUCAACAAAAAGUGAAAU A-108435.2 AUUUCACUUUUUGUUGAAGUAGA 447-469AD-52693.1 A-108889.1 AGAGCAACUAACUAACUUAAU A-108451.2AUUAAGUUAGUUAGUUGCUCUUC 475-497 AD-52694.1 A-108897.1GAUAAUAGCAUCAAAGACCUU A-108467.2 AAGGUCUUUGAUGCUAUUAUCUU 563-585AD-52695.1 A-108905.1 CAUAGUCAAAUAAAAGAAAUA A-108483.2UAUUUCUUUUAUUUGACUAUGCU 626-648 AD-52696.1 A-108866.1UAUAAACUACAAGUCAAAAAU A-108405.2 AUUUUUGACUUGUAGUUUAUAUG 359-381AD-52697.1 A-108874.1 AACUCAACUCAAAACUUGAAA A-108421.2UUUCAAGUUUUGAGUUGAGUUCA 405-427 AD-52698.1 A-108882.1ACUUCAACAAAAAGUGAAAUA A-108437.2 UAUUUCACUUUUUGUUGAAGUAG 448-470AD-52699.1 A-108890.1 GAGCAACUAACUAACUUAAUU A-108453.2AAUUAAGUUAGUUAGUUGCUCUU 476-498 AD-52700.1 A-108898.1AACCAACAGCAUAGUCAAAUA A-108469.2 UAUUUGACUAUGCUGUUGGUUUA 617-639AD-52701.1 A-108906.1 AGUCAAAUAAAAGAAAUAGAA A-108485.2UUCUAUUUCUUUUAUUUGACUAU 629-651 AD-52702.1 A-108867.1AGUCAAAAAUGAAGAGGUAAA A-108407.2 UUUACCUCUUCAUUUUUGACUUG 370-392AD-52703.1 A-108875.1 CUUGAAAGCCUCCUAGAAGAA A-108423.2UUCUUCUAGGAGGCUUUCAAGUU 419-441 AD-52704.1 A-108883.1CUUCAACAAAAAGUGAAAUAU A-108439.2 AUAUUUCACUUUUUGUUGAAGUA 449-471AD-52705.1 A-108891.1 CAACUAACUAACUUAAUUCAA A-108455.2UUGAAUUAAGUUAGUUAGUUGCU 479-501 AD-52706.1 A-108899.1ACCAACAGCAUAGUCAAAUAA A-108471.2 UUAUUUGACUAUGCUGUUGGUUU 618-640AD-52707.1 A-108907.1 GAACCCACAGAAAUUUCUCUA A-108487.2UAGAGAAAUUUCUGUGGGUUCUU 677-699 AD-52708.1 A-108868.1GAAUAUGUCACUUGAACUCAA A-108409.2 UUGAGUUCAAGUGACAUAUUCUU 391-413AD-52709.1 A-108876.1 UGAAAGCCUCCUAGAAGAAAA A-108425.2UUUUCUUCUAGGAGGCUUUCAAG 421-443 AD-52710.1 A-108884.1UUCAACAAAAAGUGAAAUAUU A-108441.2 AAUAUUUCACUUUUUGUUGAAGU 450-472AD-52711.1 A-108892.1 AACUAACUAACUUAAUUCAAA A-108457.2UUUGAAUUAAGUUAGUUAGUUGC 480-502 AD-52712.1 A-108900.1CCAACAGCAUAGUCAAAUAAA A-108473.2 UUUAUUUGACUAUGCUGUUGGUU 619-641AD-52713.1 A-108908.1 AACCCACAGAAAUUUCUCUAU A-108489.2AUAGAGAAAUUUCUGUGGGUUCU 678-700 AD-52714.1 A-108869.1UGUCACUUGAACUCAACUCAA A-108411.2 UUGAGUUGAGUUCAAGUGACAUA 396-418AD-52715.1 A-108877.1 GAAAGCCUCCUAGAAGAAAAA A-108427.2UUUUUCUUCUAGGAGGCUUUCAA 422-444 AD-52716.1 A-108885.1AAUAUUUAGAAGAGCAACUAA A-108443.2 UUAGUUGCUCUUCUAAAUAUUUC 465-487AD-52717.1 A-108893.1 ACUAACUAACUUAAUUCAAAA A-108459.2UUUUGAAUUAAGUUAGUUAGUUG 481-503 AD-52718.1 A-108901.1CAACAGCAUAGUCAAAUAAAA A-108475.2 UUUUAUUUGACUAUGCUGUUGGU 620-642AD-52719.1 A-108909.1 CCACAGAAAUUUCUCUAUCUU A-108491.2AAGAUAGAGAAAUUUCUGUGGGU 681-703 AD-52720.1 A-108870.1GUCACUUGAACUCAACUCAAA A-108413.2 UUUGAGUUGAGUUCAAGUGACAU 397-419AD-52721.1 A-108878.1 CUCCUAGAAGAAAAAAUUCUA A-108429.2UAGAAUUUUUUCUUCUAGGAGGC 428-450 AD-52722.1 A-108886.1AUUUAGAAGAGCAACUAACUA A-108445.2 UAGUUAGUUGCUCUUCUAAAUAU 468-490AD-52723.1 A-108894.1 CUAACUAACUUAAUUCAAAAU A-108461.2AUUUUGAAUUAAGUUAGUUAGUU 482-504 AD-52724.1 A-108902.1CAGCAUAGUCAAAUAAAAGAA A-108477.2 UUCUUUUAUUUGACUAUGCUGUU 623-645AD-52725.1 A-108910.1 GAAAUAAGAAAUGUAAAACAU A-108493.2AUGUUUUACAUUUCUUAUUUCAU 746-768 AD-52726.1 A-108871.1UCACUUGAACUCAACUCAAAA A-108415.2 UUUUGAGUUGAGUUCAAGUGACA 398-420AD-52727.1 A-108879.1 UCUACUUCAACAAAAAGUGAA A-108431.2UUCACUUUUUGUUGAAGUAGAAU 445-467 AD-52728.1 A-108887.1UUUAGAAGAGCAACUAACUAA A-108447.2 UUAGUUAGUUGCUCUUCUAAAUA 469-491AD-52729.1 A-108895.1 AAAACAAGAUAAUAGCAUCAA A-108463.2UUGAUGCUAUUAUCUUGUUUUUC 556-578 AD-52730.1 A-108903.1AGCAUAGUCAAAUAAAAGAAA A-108479.2 UUUCUUUUAUUUGACUAUGCUGU 624-646AD-52731.1 A-108958.1 AGACCCAGCAACUCUCAAGUU A-108495.2AACUUGAGAGUUGCUGGGUCUGA 836-858 AD-52732.1 A-108966.1AGUCCAUGGACAUUAAUUCAA A-108511.2 UUGAAUUAAUGUCCAUGGACUAC 887-909AD-52733.1 A-108974.1 GAUGGAUCACAAAACUUCAAU A-108527.2AUUGAAGUUUUGUGAUCCAUCUA 917-939 AD-52734.1 A-108982.1CUAGAGAAGAUAUACUCCAUA A-108543.2 UAUGGAGUAUAUCUUCUCUAGGC  998-1020AD-52735.1 A-108990.1 AAAGACAACAAACAUUAUAUU A-108559.2AAUAUAAUGUUUGUUGUCUUUCC 1064-1086 AD-52736.1 A-108998.1CAUUAUAUUGAAUAUUCUUUU A-108575.2 AAAAGAAUAUUCAAUAUAAUGUU 1076-1098AD-52737.1 A-108959.1 GACCCAGCAACUCUCAAGUUU A-108497.2AAACUUGAGAGUUGCUGGGUCUG 837-859 AD-52739.1 A-108975.1GGAUCACAAAACUUCAAUGAA A-108529.2 UUCAUUGAAGUUUUGUGAUCCAU 920-942AD-52740.1 A-108983.1 GAAGAUAUACUCCAUAGUGAA A-108545.2UUCACUAUGGAGUAUAUCUUCUC 1003-1025 AD-52741.1 A-108991.1GACAACAAACAUUAUAUUGAA A-108561.2 UUCAAUAUAAUGUUUGUUGUCUU 1067-1089AD-52742.1 A-108999.1 GGGAAAUCACGAAACCAACUA A-108577.2UAGUUGGUUUCGUGAUUUCCCAA 1102-1124 AD-52743.1 A-108960.1ACCCAGCAACUCUCAAGUUUU A-108499.2 AAAACUUGAGAGUUGCUGGGUCU 838-860AD-52744.1 A-108968.1 GGACAUUAAUUCAACAUCGAA A-108515.2UUCGAUGUUGAAUUAAUGUCCAU 894-916 AD-52745.1 A-108976.1GAUCACAAAACUUCAAUGAAA A-108531.2 UUUCAUUGAAGUUUUGUGAUCCA 921-943AD-52746.1 A-108984.1 ACUCCAUAGUGAAGCAAUCUA A-108547.2UAGAUUGCUUCACUAUGGAGUAU 1011-1033 AD-52747.1 A-108992.1ACAACAAACAUUAUAUUGAAU A-108563.2 AUUCAAUAUAAUGUUUGUUGUCU 1068-1090AD-52748.1 A-109000.1 GGAAAUCACGAAACCAACUAU A-108579.2AUAGUUGGUUUCGUGAUUUCCCA 1103-1125 AD-52749.1 A-108961.1CCCAGCAACUCUCAAGUUUUU A-108501.2 AAAAACUUGAGAGUUGCUGGGUC 839-861AD-52750.1 A-108969.1 GACAUUAAUUCAACAUCGAAU A-108517.2AUUCGAUGUUGAAUUAAUGUCCA 895-917 AD-52751.1 A-108977.1AACGUGGGAGAACUACAAAUA A-108533.2 UAUUUGUAGUUCUCCCACGUUUC 940-962AD-52752.1 A-108985.1 CUCCAUAGUGAAGCAAUCUAA A-108549.2UUAGAUUGCUUCACUAUGGAGUA 1012-1034 AD-52753.1 A-108993.1CAACAAACAUUAUAUUGAAUA A-108565.2 UAUUCAAUAUAAUGUUUGUUGUC 1069-1091AD-52754.1 A-109001.1 GAAAUCACGAAACCAACUAUA A-108581.2UAUAGUUGGUUUCGUGAUUUCCC 1104-1126 AD-52755.1 A-108962.1CUCUCAAGUUUUUCAUGUCUA A-108503.2 UAGACAUGAAAAACUUGAGAGUU 847-869AD-52756.1 A-108970.1 ACAUUAAUUCAACAUCGAAUA A-108519.2UAUUCGAUGUUGAAUUAAUGUCC 896-918 AD-52757.1 A-108978.1GGGAGAACUACAAAUAUGGUU A-108535.2 AACCAUAUUUGUAGUUCUCCCAC 945-967AD-52758.1 A-108986.1 UCCAUAGUGAAGCAAUCUAAU A-108551.2AUUAGAUUGCUUCACUAUGGAGU 1013-1035 AD-52759.1 A-108994.1AACAAACAUUAUAUUGAAUAU A-108567.2 AUAUUCAAUAUAAUGUUUGUUGU 1070-1092AD-52760.1 A-109002.1 UGGCAAUGUCCCCAAUGCAAU A-108583.2AUUGCAUUGGGGACAUUGCCAGU 1147-1169 AD-52761.1 A-108963.1UCAGGUAGUCCAUGGACAUUA A-108505.2 UAAUGUCCAUGGACUACCUGAUA 881-903AD-52762.1 A-108971.1 UUAAUUCAACAUCGAAUAGAU A-108521.2AUCUAUUCGAUGUUGAAUUAAUG 899-921 AD-52763.1 A-108979.1GGAGAACUACAAAUAUGGUUU A-108537.2 AAACCAUAUUUGUAGUUCUCCCA 946-968AD-52764.1 A-108987.1 CCAUAGUGAAGCAAUCUAAUU A-108553.2AAUUAGAUUGCUUCACUAUGGAG 1014-1036 AD-52765.1 A-108995.1ACAAACAUUAUAUUGAAUAUU A-108569.2 AAUAUUCAAUAUAAUGUUUGUUG 1071-1093AD-52766.1 A-109003.1 AAUGCAAUCCCGGAAAACAAA A-108585.2UUUGUUUUCCGGGAUUGCAUUGG 1160-1182 AD-52767.1 A-108964.1CAGGUAGUCCAUGGACAUUAA A-108507.2 UUAAUGUCCAUGGACUACCUGAU 882-904AD-52768.1 A-108972.1 UUCAACAUCGAAUAGAUGGAU A-108523.2AUCCAUCUAUUCGAUGUUGAAUU 903-925 AD-52769.1 A-108980.1GUUGGGCCUAGAGAAGAUAUA A-108539.2 UAUAUCUUCUCUAGGCCCAACCA  991-1013AD-52770.1 A-108988.1 CAUAGUGAAGCAAUCUAAUUA A-108555.2UAAUUAGAUUGCUUCACUAUGGA 1015-1037 AD-52771.1 A-108996.1AACAUUAUAUUGAAUAUUCUU A-108571.2 AAGAAUAUUCAAUAUAAUGUUUG 1074-1096AD-52772.1 A-109004.1 GCAAUCCCGGAAAACAAAGAU A-108587.2AUCUUUGUUUUCCGGGAUUGCAU 1163-1185 AD-52773.1 A-108965.1GGUAGUCCAUGGACAUUAAUU A-108509.2 AAUUAAUGUCCAUGGACUACCUG 884-906AD-52774.1 A-108973.1 AUCGAAUAGAUGGAUCACAAA A-108525.2UUUGUGAUCCAUCUAUUCGAUGU 909-931 AD-52775.1 A-108981.1CCUAGAGAAGAUAUACUCCAU A-108541.2 AUGGAGUAUAUCUUCUCUAGGCC  997-1019AD-52776.1 A-108989.1 GUUGGAAGACUGGAAAGACAA A-108557.2UUGUCUUUCCAGUCUUCCAACUC 1051-1073 AD-52777.1 A-108997.1ACAUUAUAUUGAAUAUUCUUU A-108573.2 AAAGAAUAUUCAAUAUAAUGUUU 1075-1097AD-52778.1 A-109005.1 CAAUCCCGGAAAACAAAGAUU A-108589.2AAUCUUUGUUUUCCGGGAUUGCA 1164-1186 AD-52779.1 A-109013.1CUACUUGGGAUCACAAAGCAA A-108605.2 UUGCUUUGUGAUCCCAAGUAGAA 1194-1216AD-52780.1 A-109021.1 ACAACCUAAAUGGUAAAUAUA A-108621.2UAUAUUUACCAUUUAGGUUGUUU 1281-1303 AD-52781.1 A-109029.1AUCCAUCCAACAGAUUCAGAA A-108637.2 UUCUGAAUCUGUUGGAUGGAUCA 1400-1422AD-52782.1 A-109037.1 AACUGAGGCAAAUUUAAAAGA A-108653.2UCUUUUAAAUUUGCCUCAGUUCA 1432- 1454_G21A AD-52783.1 A-109045.1AGAGUAUGUGUAAAAAUCUGU A-108669.2 ACAGAUUUUUACACAUACUCUGU 1913-1935AD-52784.1 A-109006.1 AAUCCCGGAAAACAAAGAUUU A-108591.2AAAUCUUUGUUUUCCGGGAUUGC 1165-1187 AD-52785.1 A-109014.1UACUUGGGAUCACAAAGCAAA A-108607.2 UUUGCUUUGUGAUCCCAAGUAGA 1195-1217AD-52786.1 A-109022.1 CAACCUAAAUGGUAAAUAUAA A-108623.2UUAUAUUUACCAUUUAGGUUGUU 1282-1304 AD-52787.1 A-109030.1UUGAAUGAACUGAGGCAAAUU A-108639.2 AAUUUGCCUCAGUUCAUUCAAAG 1425-1447AD-52788.1 A-109038.1 ACUGAGGCAAAUUUAAAAGGA A-108655.2UCCUUUUAAAUUUGCCUCAGUUC 1433- 1455_C21A AD-52789.1 A-109046.1GAGUAUGUGUAAAAAUCUGUA A-108671.2 UACAGAUUUUUACACAUACUCUG 1914-1936AD-52791.1 A-109015.1 ACUUGGGAUCACAAAGCAAAA A-108609.2UUUUGCUUUGUGAUCCCAAGUAG 1196-1218 AD-52792.1 A-109023.1AUGGUAAAUAUAACAAACCAA A-108625.2 UUGGUUUGUUAUAUUUACCAUUU 1290-1312AD-52793.1 A-109031.1 UGAAUGAACUGAGGCAAAUUU A-108641.2AAAUUUGCCUCAGUUCAUUCAAA 1426-1448 AD-52794.1 A-109039.1CUGAGGCAAAUUUAAAAGGCA A-108657.2 UGCCUUUUAAAUUUGCCUCAGUU 1434-1456AD-52795.1 A-109047.1 AGUAUGUGUAAAAAUCUGUAA A-108673.2UUACAGAUUUUUACACAUACUCU 1915-1937 AD-52796.1 A-109008.1GAAAACAAAGAUUUGGUGUUU A-108595.2 AAACACCAAAUCUUUGUUUUCCG 1172-1194AD-52797.1 A-109016.1 AGUGUGGAGAAAACAACCUAA A-108611.2UUAGGUUGUUUUCUCCACACUCA 1269-1291 AD-52798.1 A-109024.1GUCUCAAAAUGGAAGGUUAUA A-108627.2 UAUAACCUUCCAUUUUGAGACUU 1354-1376AD-52799.1 A-109032.1 GAAUGAACUGAGGCAAAUUUA A-108643.2UAAAUUUGCCUCAGUUCAUUCAA 1427-1449 AD-52800.1 A-109040.1UGAGGCAAAUUUAAAAGGCAA A-108659.2 UUGCCUUUUAAAUUUGCCUCAGU 1435-1457AD-52801.1 A-109048.1 GUAUGUGUAAAAAUCUGUAAU A-108675.2AUUACAGAUUUUUACACAUACUC 1916-1938 AD-52802.1 A-109009.1AAAACAAAGAUUUGGUGUUUU A-108597.2 AAAACACCAAAUCUUUGUUUUCC 1173-1195AD-52803.1 A-109017.1 GUGUGGAGAAAACAACCUAAA A-108613.2UUUAGGUUGUUUUCUCCACACUC 1270-1292 AD-52804.1 A-109025.1AUGGAAGGUUAUACUCUAUAA A-108629.2 UUAUAGAGUAUAACCUUCCAUUU 1362-1384AD-52805.1 A-109033.1 AAUGAACUGAGGCAAAUUUAA A-108645.2UUAAAUUUGCCUCAGUUCAUUCA 1428-1450 AD-52806.1 A-109041.1GAGGCAAAUUUAAAAGGCAAU A-108661.2 AUUGCCUUUUAAAUUUGCCUCAG 1436-1458AD-52807.1 A-109049.1 UAUGUGUAAAAAUCUGUAAUA A-108677.2UAUUACAGAUUUUUACACAUACU 1917-1939 AD-52808.1 A-109010.1ACAAAGAUUUGGUGUUUUCUA A-108599.2 UAGAAAACACCAAAUCUUUGUUU 1176-1198AD-52809.1 A-109018.1 UGUGGAGAAAACAACCUAAAU A-108615.2AUUUAGGUUGUUUUCUCCACACU 1271-1293 AD-52810.1 A-109026.1UGGAAGGUUAUACUCUAUAAA A-108631.2 UUUAUAGAGUAUAACCUUCCAUU 1363-1385AD-52811.1 A-109034.1 AUGAACUGAGGCAAAUUUAAA A-108647.2UUUAAAUUUGCCUCAGUUCAUUC 1429-1451 AD-52812.1 A-109042.1AGGCAAAUUUAAAAGGCAAUA A-108663.2 UAUUGCCUUUUAAAUUUGCCUCA 1437-1459AD-52813.1 A-109011.1 AAGAUUUGGUGUUUUCUACUU A-108601.2AAGUAGAAAACACCAAAUCUUUG 1179-1201 AD-52814.1 A-109019.1AAACAACCUAAAUGGUAAAUA A-108617.2 UAUUUACCAUUUAGGUUGUUUUC 1279-1301AD-52815.1 A-109027.1 AUACUCUAUAAAAUCAACCAA A-108633.2UUGGUUGAUUUUAUAGAGUAUAA 1372-1394 AD-52816.1 A-109035.1UGAACUGAGGCAAAUUUAAAA A-108649.2 UUUUAAAUUUGCCUCAGUUCAUU 1430-1452AD-52817.1 A-109043.1 GGCAAAUUUAAAAGGCAAUAA A-108665.2UUAUUGCCUUUUAAAUUUGCCUC 1438-1460 AD-52818.1 A-109012.1UUUUCUACUUGGGAUCACAAA A-108603.2 UUUGUGAUCCCAAGUAGAAAACA 1190-1212AD-52819.1 A-109020.1 AACAACCUAAAUGGUAAAUAU A-108619.2AUAUUUACCAUUUAGGUUGUUUU 1280-1302 AD-52820.1 A-109028.1UACUCUAUAAAAUCAACCAAA A-108635.2 UUUGGUUGAUUUUAUAGAGUAUA 1373-1395AD-52821.1 A-109036.1 GAACUGAGGCAAAUUUAAAAA A-108651.2UUUUUAAAUUUGCCUCAGUUCAU 1431- 1453_G21A AD-52822.1 A-109044.1CAGAGUAUGUGUAAAAAUCUU A-108667.2 AAGAUUUUUACACAUACUCUGUG 1912- 1934_G21U

TABLE 10 Modified Sense and antisense strandsequences of ANGPTL3 dsRNAs without GalNal conjugationThese sequences are the same as the sequences listedin Table 8 except that they do not contain GalNal conjugation.Sense Sequence Duplex Sense Oligo (SEQ ID NOS 1366-1546, respectively,Name Name in order of appearance) AD-52637.1 A-108817.1UfcAfcAfaUfuAfAfGfcUfcCfuUfcUfuUf AD-52638.1 A-108825.1UfuAfuUfgUfuCfCfUfcUfaGfuUfaUfuUf AD-52639.1 A-108833.1GfcUfaUfgUfuAfGfAfcGfaUfgUfaAfaAf AD-52640.1 A-108841.1GfgAfcAfuGfgUfCfUfuAfaAfgAfcUfuUf AD-52641.1 A-108849.1CfaAfaAfaCfuCfAfAfcAfuAfuUfuGfaUf AD-52642.1 A-108857.1AfcCfaGfuGfaAfAfUfcAfaAfgAfaGfaAf AD-52643.1 A-108818.1CfaCfaAfuUfaAfGfCfuCfcUfuCfuUfuUf AD-52645.1 A-108834.1CfuAfuGfuUfaGfAfCfgAfuGfuAfaAfaAf AD-52647.1 A-108850.1UfcAfaCfaUfaUfUfUfgAfuCfaGfuCfuUf AD-52648.1 A-108858.1AfaCfuGfaGfaAfGfAfaCfuAfcAfuAfuAf AD-52649.1 A-108819.1AfcAfaUfuAfaGfCfUfcCfuUfcUfuUfuUf AD-52650.1 A-108827.1CfuCfcAfgAfgCfCfAfaAfaUfcAfaGfaUf AD-52651.1 A-108835.1CfgAfuGfuAfaAfAfAfuUfuUfaGfcCfaAf AD-52652.1 A-108843.1GfuCfuUfaAfaGfAfCfuUfuGfuCfcAfuAf AD-52653.1 A-108851.1CfaAfcAfuAfuUfUfGfaUfcAfgUfcUfuUf AD-52654.1 A-108859.1AfcUfgAfgAfaGfAfAfcUfaCfaUfaUfaAf AD-52656.1 A-108828.1CfcAfgAfgCfcAfAfAfaUfcAfaGfaUfuUf AD-52657.1 A-108836.1GfaUfgUfaAfaAfAfUfuUfuAfgCfcAfaUf AD-52658.1 A-108844.1UfcUfuAfaAfgAfCfUfuUfgUfcCfaUfaAf AD-52659.1 A-108852.1AfaCfaUfaUfuUfGfAfuCfaGfuCfuUfuUf AD-52660.1 A-108860.1CfuGfaGfaAfgAfAfCfuAfcAfuAfuAfaAf AD-52661.1 A-108821.1AfaUfuAfaGfcUfCfCfuUfcUfuUfuUfaUf AD-52662.1 A-108829.1AfaAfuCfaAfgAfUfUfuGfcUfaUfgUfuAf AD-52663.1 A-108837.1UfuCfaGfuUfgGfGfAfcAfuGfgUfcUfuAf AD-52664.1 A-108845.1GfgGfcCfaAfaUfUfAfaUfgAfcAfuAfuUf AD-52665.1 A-108853.1AfcAfuAfuUfuGfAfUfcAfgUfcUfuUfuUf AD-52666.1 A-108861.1AfgAfaCfuAfcAfUfAfuAfaAfcUfaCfaAf AD-52667.1 A-108822.1AfuUfaAfgCfuCfCfUfuCfuUfuUfuAfuUf AD-52668.1 A-108830.1AfgAfuUfuGfcUfAfUfgUfuAfgAfcGfaUf AD-52669.1 A-108838.1UfcAfgUfuGfgGfAfCfaUfgGfuCfuUfaAf AD-52670.1 A-108846.1GfgCfcAfaAfuUfAfAfuGfaCfaUfaUfuUf AD-52671.1 A-108854.1CfaUfaUfuUfgAfUfCfaGfuCfuUfuUfuAf AD-52672.1 A-108862.1UfaCfaUfaUfaAfAfCfuAfcAfaGfuCfaAf AD-52673.1 A-108823.1UfuUfuAfuUfgUfUfCfcUfcUfaGfuUfaUf AD-52674.1 A-108831.1UfuGfcUfaUfgUfUfAfgAfcGfaUfgUfaAf AD-52675.1 A-108839.1CfaGfuUfgGfgAfCfAfuGfgUfcUfuAfaAf AD-52676.1 A-108847.1AfaAfuUfaAfuGfAfCfaUfaUfuUfcAfaAf AD-52677.1 A-108855.1GfaUfcAfgUfcUfUfUfuUfaUfgAfuCfuAf AD-52678.1 A-108863.1AfcAfuAfuAfaAfCfUfaCfaAfgUfcAfaAf AD-52679.1 A-108824.1UfuUfaUfuGfuUfCfCfuCfuAfgUfuAfuUf AD-52680.1 A-108832.1UfgCfuAfuGfuUfAfGfaCfgAfuGfuAfaAf AD-52681.1 A-108840.1GfgGfaCfaUfgGfUfCfuUfaAfaGfaCfuUf AD-52682.1 A-108848.1UfgAfcAfuAfuUfUfCfaAfaAfaCfuCfaAf AD-52683.1 A-108856.1AfuCfaGfuCfuUfUfUfuAfuGfaUfcUfaUf AD-52684.1 A-108864.1CfaUfaUfaAfaCfUfAfcAfaGfuCfaAfaAf AD-52685.1 A-108872.1CfuUfgAfaCfuCfAfAfcUfcAfaAfaCfuUf AD-52686.1 A-108880.1CfuAfcUfuCfaAfCfAfaAfaAfgUfgAfaAf AD-52687.1 A-108888.1AfaGfaGfcAfaCfUfAfaCfuAfaCfuUfaAf AD-52688.1 A-108896.1AfaAfcAfaGfaUfAfAfuAfgCfaUfcAfaAf AD-52689.1 A-108904.1GfcAfuAfgUfcAfAfAfuAfaAfaGfaAfaUf AD-52690.1 A-108865.1AfuAfuAfaAfcUfAfCfaAfgUfcAfaAfaAf AD-52691.1 A-108873.1GfaAfcUfcAfaCfUfCfaAfaAfcUfuGfaAf AD-52692.1 A-108881.1UfaCfuUfcAfaCfAfAfaAfaGfuGfaAfaUf AD-52693.1 A-108889.1AfgAfgCfaAfcUfAfAfcUfaAfcUfuAfaUf AD-52694.1 A-108897.1GfaUfaAfuAfgCfAfUfcAfaAfgAfcCfuUf AD-52695.1 A-108905.1CfaUfaGfuCfaAfAfUfaAfaAfgAfaAfuAf AD-52696.1 A-108866.1UfaUfaAfaCfuAfCfAfaGfuCfaAfaAfaUf AD-52697.1 A-108874.1AfaCfuCfaAfcUfCfAfaAfaCfuUfgAfaAf AD-52698.1 A-108882.1AfcUfuCfaAfcAfAfAfaAfgUfgAfaAfuAf AD-52699.1 A-108890.1GfaGfcAfaCfuAfAfCfuAfaCfuUfaAfuUf AD-52700.1 A-108898.1AfaCfcAfaCfaGfCfAfuAfgUfcAfaAfuAf AD-52701.1 A-108906.1AfgUfcAfaAfuAfAfAfaGfaAfaUfaGfaAf AD-52702.1 A-108867.1AfgUfcAfaAfaAfUfGfaAfgAfgGfuAfaAf AD-52703.1 A-108875.1CfuUfgAfaAfgCfCfUfcCfuAfgAfaGfaAf AD-52704.1 A-108883.1CfuUfcAfaCfaAfAfAfaGfuGfaAfaUfaUf AD-52705.1 A-108891.1CfaAfcUfaAfcUfAfAfcUfuAfaUfuCfaAf AD-52706.1 A-108899.1AfcCfaAfcAfgCfAfUfaGfuCfaAfaUfaAf AD-52707.1 A-108907.1GfaAfcCfcAfcAfGfAfaAfuUfuCfuCfuAf AD-52708.1 A-108868.1GfaAfuAfuGfuCfAfCfuUfgAfaCfuCfaAf AD-52709.1 A-108876.1UfgAfaAfgCfcUfCfCfuAfgAfaGfaAfaAf AD-52710.1 A-108884.1UfuCfaAfcAfaAfAfAfgUfgAfaAfuAfuUf AD-52711.1 A-108892.1AfaCfuAfaCfuAfAfCfuUfaAfuUfcAfaAf AD-52712.1 A-108900.1CfcAfaCfaGfcAfUfAfgUfcAfaAfuAfaAf AD-52713.1 A-108908.1AfaCfcCfaCfaGfAfAfaUfuUfcUfcUfaUf AD-52714.1 A-108869.1UfgUfcAfcUfuGfAfAfcUfcAfaCfuCfaAf AD-52715.1 A-108877.1GfaAfaGfcCfuCfCfUfaGfaAfgAfaAfaAf AD-52716.1 A-108885.1AfaUfaUfuUfaGfAfAfgAfgCfaAfcUfaAf AD-52717.1 A-108893.1AfcUfaAfcUfaAfCfUfuAfaUfuCfaAfaAf AD-52718.1 A-108901.1CfaAfcAfgCfaUfAfGfuCfaAfaUfaAfaAf AD-52719.1 A-108909.1CfcAfcAfgAfaAfUfUfuCfuCfuAfuCfuUf AD-52720.1 A-108870.1GfuCfaCfuUfgAfAfCfuCfaAfcUfcAfaAf AD-52721.1 A-108878.1CfuCfcUfaGfaAfGfAfaAfaAfaUfuCfuAf AD-52722.1 A-108886.1AfuUfuAfgAfaGfAfGfcAfaCfuAfaCfuAf AD-52723.1 A-108894.1CfuAfaCfuAfaCfUfUfaAfuUfcAfaAfaUf AD-52724.1 A-108902.1CfaGfcAfuAfgUfCfAfaAfuAfaAfaGfaAf AD-52725.1 A-108910.1GfaAfaUfaAfgAfAfAfuGfuAfaAfaCfaUf AD-52726.1 A-108871.1UfcAfcUfuGfaAfCfUfcAfaCfuCfaAfaAf AD-52727.1 A-108879.1UfcUfaCfuUfcAfAfCfaAfaAfaGfuGfaAf AD-52728.1 A-108887.1UfuUfaGfaAfgAfGfCfaAfcUfaAfcUfaAf AD-52729.1 A-108895.1AfaAfaCfaAfgAfUfAfaUfaGfcAfuCfaAf AD-52730.1 A-108903.1AfgCfaUfaGfuCfAfAfaUfaAfaAfgAfaAf AD-52731.1 A-108958.1AfgAfcCfcAfgCfAfAfcUfcUfcAfaGfuUf AD-52732.1 A-108966.1AfgUfcCfaUfgGfAfCfaUfuAfaUfuCfaAf AD-52733.1 A-108974.1GfaUfgGfaUfcAfCfAfaAfaCfuUfcAfaUf AD-52734.1 A-108982.1CfuAfgAfgAfaGfAfUfaUfaCfuCfcAfuAf AD-52735.1 A-108990.1AfaAfgAfcAfaCfAfAfaCfaUfuAfuAfuUf AD-52736.1 A-108998.1CfaUfuAfuAfuUfGfAfaUfaUfuCfuUfuUf AD-52737.1 A-108959.1GfaCfcCfaGfcAfAfCfuCfuCfaAfgUfuUf AD-52739.1 A-108975.1GfgAfuCfaCfaAfAfAfcUfuCfaAfuGfaAf AD-52740.1 A-108983.1GfaAfgAfuAfuAfCfUfcCfaUfaGfuGfaAf AD-52741.1 A-108991.1GfaCfaAfcAfaAfCfAfuUfaUfaUfuGfaAf AD-52742.1 A-108999.1GfgGfaAfaUfcAfCfGfaAfaCfcAfaCfuAf AD-52743.1 A-108960.1AfcCfcAfgCfaAfCfUfcUfcAfaGfuUfuUf AD-52744.1 A-108968.1GfgAfcAfuUfaAfUfUfcAfaCfaUfcGfaAf AD-52745.1 A-108976.1GfaUfcAfcAfaAfAfCfuUfcAfaUfgAfaAf AD-52746.1 A-108984.1AfcUfcCfaUfaGfUfGfaAfgCfaAfuCfuAf AD-52747.1 A-108992.1AfcAfaCfaAfaCfAfUfuAfuAfuUfgAfaUf AD-52748.1 A-109000.1GfgAfaAfuCfaCfGfAfaAfcCfaAfcUfaUf AD-52749.1 A-108961.1CfcCfaGfcAfaCfUfCfuCfaAfgUfuUfuUf AD-52750.1 A-108969.1GfaCfaUfuAfaUfUfCfaAfcAfuCfgAfaUf AD-52751.1 A-108977.1AfaCfgUfgGfgAfGfAfaCfuAfcAfaAfuAf AD-52752.1 A-108985.1CfuCfcAfuAfgUfGfAfaGfcAfaUfcUfaAf AD-52753.1 A-108993.1CfaAfcAfaAfcAfUfUfaUfaUfuGfaAfuAf AD-52754.1 A-109001.1GfaAfaUfcAfcGfAfAfaCfcAfaCfuAfuAf AD-52755.1 A-108962.1CfuCfuCfaAfgUfUfUfuUfcAfuGfuCfuAf AD-52756.1 A-108970.1AfcAfuUfaAfuUfCfAfaCfaUfcGfaAfuAf AD-52757.1 A-108978.1GfgGfaGfaAfcUfAfCfaAfaUfaUfgGfuUf AD-52758.1 A-108986.1UfcCfaUfaGfuGfAfAfgCfaAfuCfuAfaUf AD-52759.1 A-108994.1AfaCfaAfaCfaUfUfAfuAfuUfgAfaUfaUf AD-52760.1 A-109002.1UfgGfcAfaUfgUfCfCfcCfaAfuGfcAfaUf AD-52761.1 A-108963.1UfcAfgGfuAfgUfCfCfaUfgGfaCfaUfuAf AD-52762.1 A-108971.1UfuAfaUfuCfaAfCfAfuCfgAfaUfaGfaUf AD-52763.1 A-108979.1GfgAfgAfaCfuAfCfAfaAfuAfuGfgUfuUf AD-52764.1 A-108987.1CfcAfuAfgUfgAfAfGfcAfaUfcUfaAfuUf AD-52765.1 A-108995.1AfcAfaAfcAfuUfAfUfaUfuGfaAfuAfuUf AD-52766.1 A-109003.1AfaUfgCfaAfuCfCfCfgGfaAfaAfcAfaAf AD-52767.1 A-108964.1CfaGfgUfaGfuCfCfAfuGfgAfcAfuUfaAf AD-52768.1 A-108972.1UfuCfaAfcAfuCfGfAfaUfaGfaUfgGfaUf AD-52769.1 A-108980.1GfuUfgGfgCfcUfAfGfaGfaAfgAfuAfuAf AD-52770.1 A-108988.1CfaUfaGfuGfaAfGfCfaAfuCfuAfaUfuAf AD-52771.1 A-108996.1AfaCfaUfuAfuAfUfUfgAfaUfaUfuCfuUf AD-52772.1 A-109004.1GfcAfaUfcCfcGfGfAfaAfaCfaAfaGfaUf AD-52773.1 A-108965.1GfgUfaGfuCfcAfUfGfgAfcAfuUfaAfuUf AD-52774.1 A-108973.1AfuCfgAfaUfaGfAfUfgGfaUfcAfcAfaAf AD-52775.1 A-108981.1CfcUfaGfaGfaAfGfAfuAfuAfcUfcCfaUf AD-52776.1 A-108989.1GfuUfgGfaAfgAfCfUfgGfaAfaGfaCfaAf AD-52777.1 A-108997.1AfcAfuUfaUfaUfUfGfaAfuAfuUfcUfuUf AD-52778.1 A-109005.1CfaAfuCfcCfgGfAfAfaAfcAfaAfgAfuUf AD-52779.1 A-109013.1CfuAfcUfuGfgGfAfUfcAfcAfaAfgCfaAf AD-52780.1 A-109021.1AfcAfaCfcUfaAfAfUfgGfuAfaAfuAfuAf AD-52781.1 A-109029.1AfuCfcAfuCfcAfAfCfaGfaUfuCfaGfaAf AD-52782.1 A-109037.1AfaCfuGfaGfgCfAfAfaUfuUfaAfaAfgAf AD-52783.1 A-109045.1AfgAfgUfaUfgUfGfUfaAfaAfaUfcUfgUf AD-52784.1 A-109006.1AfaUfcCfcGfgAfAfAfaCfaAfaGfaUfuUf AD-52785.1 A-109014.1UfaCfuUfgGfgAfUfCfaCfaAfaGfcAfaAf AD-52786.1 A-109022.1CfaAfcCfuAfaAfUfGfgUfaAfaUfaUfaAf AD-52787.1 A-109030.1UfuGfaAfuGfaAfCfUfgAfgGfcAfaAfuUf AD-52788.1 A-109038.1AfcUfgAfgGfcAfAfAfuUfuAfaAfaGfgAf AD-52789.1 A-109046.1GfaGfuAfuGfuGfUfAfaAfaAfuCfuGfuAf AD-52791.1 A-109015.1AfcUfuGfgGfaUfCfAfcAfaAfgCfaAfaAf AD-52792.1 A-109023.1AfuGfgUfaAfaUfAfUfaAfcAfaAfcCfaAf AD-52793.1 A-109031.1UfgAfaUfgAfaCfUfGfaGfgCfaAfaUfuUf AD-52794.1 A-109039.1CfuGfaGfgCfaAfAfUfuUfaAfaAfgGfcAf AD-52795.1 A-109047.1AfgUfaUfgUfgUfAfAfaAfaUfcUfgUfaAf AD-52796.1 A-109008.1GfaAfaAfcAfaAfGfAfuUfuGfgUfgUfuUf AD-52797.1 A-109016.1AfgUfgUfgGfaGfAfAfaAfcAfaCfcUfaAf AD-52798.1 A-109024.1GfuCfuCfaAfaAfUfGfgAfaGfgUfuAfuAf AD-52799.1 A-109032.1GfaAfuGfaAfcUfGfAfgGfcAfaAfuUfuAf AD-52800.1 A-109040.1UfgAfgGfcAfaAfUfUfuAfaAfaGfgCfaAf AD-52801.1 A-109048.1GfuAfuGfuGfuAfAfAfaAfuCfuGfuAfaUf AD-52802.1 A-109009.1AfaAfaCfaAfaGfAfUfuUfgGfuGfuUfuUf AD-52803.1 A-109017.1GfuGfuGfgAfgAfAfAfaCfaAfcCfuAfaAf AD-52804.1 A-109025.1AfuGfgAfaGfgUfUfAfuAfcUfcUfaUfaAf AD-52805.1 A-109033.1AfaUfgAfaCfuGfAfGfgCfaAfaUfuUfaAf AD-52806.1 A-109041.1GfaGfgCfaAfaUfUfUfaAfaAfgGfcAfaUf AD-52807.1 A-109049.1UfaUfgUfgUfaAfAfAfaUfcUfgUfaAfuAf AD-52808.1 A-109010.1AfcAfaAfgAfuUfUfGfgUfgUfuUfuCfuAf AD-52809.1 A-109018.1UfgUfgGfaGfaAfAfAfcAfaCfcUfaAfaUf AD-52810.1 A-109026.1UfgGfaAfgGfuUfAfUfaCfuCfuAfuAfaAf AD-52811.1 A-109034.1AfuGfaAfcUfgAfGfGfcAfaAfuUfuAfaAf AD-52812.1 A-109042.1AfgGfcAfaAfuUfUfAfaAfaGfgCfaAfuAf AD-52813.1 A-109011.1AfaGfaUfuUfgGfUfGfuUfuUfcUfaCfuUf AD-52814.1 A-109019.1AfaAfcAfaCfcUfAfAfaUfgGfuAfaAfuAf AD-52815.1 A-109027.1AfuAfcUfcUfaUfAfAfaAfuCfaAfcCfaAf AD-52816.1 A-109035.1UfgAfaCfuGfaGfGfCfaAfaUfuUfaAfaAf AD-52817.1 A-109043.1GfgCfaAfaUfuUfAfAfaAfgGfcAfaUfaAf AD-52818.1 A-109012.1UfuUfuCfuAfcUfUfGfgGfaUfcAfcAfaAf AD-52819.1 A-109020.1AfaCfaAfcCfuAfAfAfuGfgUfaAfaUfaUf AD-52820.1 A-109028.1UfaCfuCfuAfuAfAfAfaUfcAfaCfcAfaAf AD-52821.1 A-109036.1GfaAfcUfgAfgGfCfAfaAfuUfuAfaAfaAf AD-52822.1 A-109044.1CfaGfaGfuAfuGfUfGfuAfaAfaAfuCfuUf Antisense Oligo Sequence DuplexAntisense (SEQ ID NOS 1547-1727, respectively, Name OligoNamein order of appearance) AD-52637.1 A-108307.2aAfaGfaAfgGfaGfcuuAfaUfuGfuGfasAfsc AD-52638.1 A-108323.2aAfaUfaAfcUfaGfaggAfaCfaAfuAfasAfsa AD-52639.1 A-108339.2uUfuUfaCfaUfcGfucuAfaCfaUfaGfcsAfsa AD-52640.1 A-108355.2aAfaGfuCfuUfuAfagaCfcAfuGfuCfcsCfsa AD-52641.1 A-108371.2aUfcAfaAfuAfuGfuugAfgUfuUfuUfgsAfsa AD-52642.1 A-108387.2uUfcUfuCfuUfuGfauuUfcAfcUfgGfusUfsu AD-52643.1 A-108309.2aAfaAfgAfaGfgAfgcuUfaAfuUfgUfgsAfsa AD-52645.1 A-108341.2uUfuUfuAfcAfuCfgucUfaAfcAfuAfgsCfsa AD-52647.1 A-108373.2aAfgAfcUfgAfuCfaaaUfaUfgUfuGfasGfsu AD-52648.1 A-108389.2uAfuAfuGfuAfgUfucuUfcUfcAfgUfusCfsc AD-52649.1 A-108311.2aAfaAfaGfaAfgGfagcUfuAfaUfuGfusGfsa AD-52650.1 A-108327.2aUfcUfuGfaUfuUfuggCfuCfuGfgAfgsAfsu AD-52651.1 A-108343.2uUfgGfcUfaAfaAfuuuUfuAfcAfuCfgsUfsc AD-52652.1 A-108359.2uAfuGfgAfcAfaAfgucUfuUfaAfgAfcsCfsa AD-52653.1 A-108375.2aAfaGfaCfuGfaUfcaaAfuAfuGfuUfgsAfsg AD-52654.1 A-108391.2uUfaUfaUfgUfaGfuucUfuCfuCfaGfusUfsc AD-52656.1 A-108329.2aAfaUfcUfuGfaUfuuuGfgCfuCfuGfgsAfsg AD-52657.1 A-108345.2aUfuGfgCfuAfaAfauuUfuUfaCfaUfcsGfsu AD-52658.1 A-108361.2uUfaUfgGfaCfaAfaguCfuUfuAfaGfasCfsc AD-52659.1 A-108377.2aAfaAfgAfcUfgAfucaAfaUfaUfgUfusGfsa AD-52660.1 A-108393.2uUfuAfuAfuGfuAfguuCfuUfcUfcAfgsUfsu AD-52661.1 A-108315.2aUfaAfaAfaGfaAfggaGfcUfuAfaUfusGfsu AD-52662.1 A-108331.2uAfaCfaUfaGfcAfaauCfuUfgAfuUfusUfsg AD-52663.1 A-108347.2uAfaGfaCfcAfuGfuccCfaAfcUfgAfasGfsg AD-52664.1 A-108363.2aAfuAfuGfuCfaUfuaaUfuUfgGfcCfcsUfsu AD-52665.1 A-108379.2aAfaAfaGfaCfuGfaucAfaAfuAfuGfusUfsg AD-52666.1 A-108395.2uUfgUfaGfuUfuAfuauGfuAfgUfuCfusUfsc AD-52667.1 A-108317.2aAfuAfaAfaAfgAfaggAfgCfuUfaAfusUfsg AD-52668.1 A-108333.2aUfcGfuCfuAfaCfauaGfcAfaAfuCfusUfsg AD-52669.1 A-108349.2uUfaAfgAfcCfaUfgucCfcAfaCfuGfasAfsg AD-52670.1 A-108365.2aAfaUfaUfgUfcAfuuaAfuUfuGfgCfcsCfsu AD-52671.1 A-108381.2uAfaAfaAfgAfcUfgauCfaAfaUfaUfgsUfsu AD-52672.1 A-108397.2uUfgAfcUfuGfuAfguuUfaUfaUfgUfasGfsu AD-52673.1 A-108319.2aUfaAfcUfaGfaGfgaaCfaAfuAfaAfasAfsg AD-52674.1 A-108335.2uUfaCfaUfcGfuCfuaaCfaUfaGfcAfasAfsu AD-52675.1 A-108351.2uUfuAfaGfaCfcAfuguCfcCfaAfcUfgsAfsa AD-52676.1 A-108367.2uUfuGfaAfaUfaUfgucAfuUfaAfuUfusGfsg AD-52677.1 A-108383.2uAfgAfuCfaUfaAfaaaGfaCfuGfaUfcsAfsa AD-52678.1 A-108399.2uUfuGfaCfuUfgUfaguUfuAfuAfuGfusAfsg AD-52679.1 A-108321.2aAfuAfaCfuAfgAfggaAfcAfaUfaAfasAfsa AD-52680.1 A-108337.2uUfuAfcAfuCfgUfcuaAfcAfuAfgCfasAfsa AD-52681.1 A-108353.2aAfgUfcUfuUfaAfgacCfaUfgUfcCfcsAfsa AD-52682.1 A-108369.2uUfgAfgUfuUfuUfgaaAfuAfuGfuCfasUfsu AD-52683.1 A-108385.2aUfaGfaUfcAfuAfaaaAfgAfcUfgAfusCfsa AD-52684.1 A-108401.2uUfuUfgAfcUfuGfuagUfuUfaUfaUfgsUfsa AD-52685.1 A-108417.2aAfgUfuUfuGfaGfuugAfgUfuCfaAfgsUfsg AD-52686.1 A-108433.2uUfuCfaCfuUfuUfuguUfgAfaGfuAfgsAfsa AD-52687.1 A-108449.2uUfaAfgUfuAfgUfuagUfuGfcUfcUfusCfsu AD-52688.1 A-108465.2uUfuGfaUfgCfuAfuuaUfcUfuGfuUfusUfsu AD-52689.1 A-108481.2aUfuUfcUfuUfuAfuuuGfaCfuAfuGfcsUfsg AD-52690.1 A-108403.2uUfuUfuGfaCfuUfguaGfuUfuAfuAfusGfsu AD-52691.1 A-108419.2uUfcAfaGfuUfuUfgagUfuGfaGfuUfcsAfsa AD-52692.1 A-108435.2aUfuUfcAfcUfuUfuugUfuGfaAfgUfasGfsa AD-52693.1 A-108451.2aUfuAfaGfuUfaGfuuaGfuUfgCfuCfusUfsc AD-52694.1 A-108467.2aAfgGfuCfuUfuGfaugCfuAfuUfaUfcsUfsu AD-52695.1 A-108483.2uAfuUfuCfuUfuUfauuUfgAfcUfaUfgsCfsu AD-52696.1 A-108405.2aUfuUfuUfgAfcUfuguAfgUfuUfaUfasUfsg AD-52697.1 A-108421.2uUfuCfaAfgUfuUfugaGfuUfgAfgUfusCfsa AD-52698.1 A-108437.2uAfuUfuCfaCfuUfuuuGfuUfgAfaGfusAfsg AD-52699.1 A-108453.2aAfuUfaAfgUfuAfguuAfgUfuGfcUfcsUfsu AD-52700.1 A-108469.2uAfuUfuGfaCfuAfugcUfgUfuGfgUfusUfsa AD-52701.1 A-108485.2uUfcUfaUfuUfcUfuuuAfuUfuGfaCfusAfsu AD-52702.1 A-108407.2uUfuAfcCfuCfuUfcauUfuUfuGfaCfusUfsg AD-52703.1 A-108423.2uUfcUfuCfuAfgGfaggCfuUfuCfaAfgsUfsu AD-52704.1 A-108439.2aUfaUfuUfcAfcUfuuuUfgUfuGfaAfgsUfsa AD-52705.1 A-108455.2uUfgAfaUfuAfaGfuuaGfuUfaGfuUfgsCfsu AD-52706.1 A-108471.2uUfaUfuUfgAfcUfaugCfuGfuUfgGfusUfsu AD-52707.1 A-108487.2uAfgAfgAfaAfuUfucuGfuGfgGfuUfcsUfsu AD-52708.1 A-108409.2uUfgAfgUfuCfaAfgugAfcAfuAfuUfcsUfsu AD-52709.1 A-108425.2uUfuUfcUfuCfuAfggaGfgCfuUfuCfasAfsg AD-52710.1 A-108441.2aAfuAfuUfuCfaCfuuuUfuGfuUfgAfasGfsu AD-52711.1 A-108457.2uUfuGfaAfuUfaAfguuAfgUfuAfgUfusGfsc AD-52712.1 A-108473.2uUfuAfuUfuGfaCfuauGfcUfgUfuGfgsUfsu AD-52713.1 A-108489.2aUfaGfaGfaAfaUfuucUfgUfgGfgUfusCfsu AD-52714.1 A-108411.2uUfgAfgUfuGfaGfuucAfaGfuGfaCfasUfsa AD-52715.1 A-108427.2uUfuUfuCfuUfcUfaggAfgGfcUfuUfcsAfsa AD-52716.1 A-108443.2uUfaGfuUfgCfuCfuucUfaAfaUfaUfusUfsc AD-52717.1 A-108459.2uUfuUfgAfaUfuAfaguUfaGfuUfaGfusUfsg AD-52718.1 A-108475.2uUfuUfaUfuUfgAfcuaUfgCfuGfuUfgsGfsu AD-52719.1 A-108491.2aAfgAfuAfgAfgAfaauUfuCfuGfuGfgsGfsu AD-52720.1 A-108413.2uUfuGfaGfuUfgAfguuCfaAfgUfgAfcsAfsu AD-52721.1 A-108429.2uAfgAfaUfuUfuUfucuUfcUfaGfgAfgsGfsc AD-52722.1 A-108445.2uAfgUfuAfgUfuGfcucUfuCfuAfaAfusAfsu AD-52723.1 A-108461.2aUfuUfuGfaAfuUfaagUfuAfgUfuAfgsUfsu AD-52724.1 A-108477.2uUfcUfuUfuAfuUfugaCfuAfuGfcUfgsUfsu AD-52725.1 A-108493.2aUfgUfuUfuAfcAfuuuCfuUfaUfuUfcsAfsu AD-52726.1 A-108415.2uUfuUfgAfgUfuGfaguUfcAfaGfuGfasCfsa AD-52727.1 A-108431.2uUfcAfcUfuUfuUfguuGfaAfgUfaGfasAfsu AD-52728.1 A-108447.2uUfaGfuUfaGfuUfgcuCfuUfcUfaAfasUfsa AD-52729.1 A-108463.2uUfgAfuGfcUfaUfuauCfuUfgUfuUfusUfsc AD-52730.1 A-108479.2uUfuCfuUfuUfaUfuugAfcUfaUfgCfusGfsu AD-52731.1 A-108495.2aAfcUfuGfaGfaGfuugCfuGfgGfuCfusGfsa AD-52732.1 A-108511.2uUfgAfaUfuAfaUfgucCfaUfgGfaCfusAfsc AD-52733.1 A-108527.2aUfuGfaAfgUfuUfuguGfaUfcCfaUfcsUfsa AD-52734.1 A-108543.2uAfuGfgAfgUfaUfaucUfuCfuCfuAfgsGfsc AD-52735.1 A-108559.2aAfuAfuAfaUfgUfuugUfuGfuCfuUfusCfsc AD-52736.1 A-108575.2aAfaAfgAfaUfaUfucaAfuAfuAfaUfgsUfsu AD-52737.1 A-108497.2aAfaCfuUfgAfgAfguuGfcUfgGfgUfcsUfsg AD-52739.1 A-108529.2uUfcAfuUfgAfaGfuuuUfgUfgAfuCfcsAfsu AD-52740.1 A-108545.2uUfcAfcUfaUfgGfaguAfuAfuCfuUfcsUfsc AD-52741.1 A-108561.2uUfcAfaUfaUfaAfuguUfuGfuUfgUfcsUfsu AD-52742.1 A-108577.2uAfgUfuGfgUfuUfcguGfaUfuUfcCfcsAfsa AD-52743.1 A-108499.2aAfaAfcUfuGfaGfaguUfgCfuGfgGfusCfsu AD-52744.1 A-108515.2uUfcGfaUfgUfuGfaauUfaAfuGfuCfcsAfsu AD-52745.1 A-108531.2uUfuCfaUfuGfaAfguuUfuGfuGfaUfcsCfsa AD-52746.1 A-108547.2uAfgAfuUfgCfuUfcacUfaUfgGfaGfusAfsu AD-52747.1 A-108563.2aUfuCfaAfuAfuAfaugUfuUfgUfuGfusCfsu AD-52748.1 A-108579.2aUfaGfuUfgGfuUfucgUfgAfuUfuCfcsCfsa AD-52749.1 A-108501.2aAfaAfaCfuUfgAfgagUfuGfcUfgGfgsUfsc AD-52750.1 A-108517.2aUfuCfgAfuGfuUfgaaUfuAfaUfgUfcsCfsa AD-52751.1 A-108533.2uAfuUfuGfuAfgUfucuCfcCfaCfgUfusUfsc AD-52752.1 A-108549.2uUfaGfaUfuGfcUfucaCfuAfuGfgAfgsUfsa AD-52753.1 A-108565.2uAfuUfcAfaUfaUfaauGfuUfuGfuUfgsUfsc AD-52754.1 A-108581.2uAfuAfgUfuGfgUfuucGfuGfaUfuUfcsCfsc AD-52755.1 A-108503.2uAfgAfcAfuGfaAfaaaCfuUfgAfgAfgsUfsu AD-52756.1 A-108519.2uAfuUfcGfaUfgUfugaAfuUfaAfuGfusCfsc AD-52757.1 A-108535.2aAfcCfaUfaUfuUfguaGfuUfcUfcCfcsAfsc AD-52758.1 A-108551.2aUfuAfgAfuUfgCfuucAfcUfaUfgGfasGfsu AD-52759.1 A-108567.2aUfaUfuCfaAfuAfuaaUfgUfuUfgUfusGfsu AD-52760.1 A-108583.2aUfuGfcAfuUfgGfggaCfaUfuGfcCfasGfsu AD-52761.1 A-108505.2uAfaUfgUfcCfaUfggaCfuAfcCfuGfasUfsa AD-52762.1 A-108521.2aUfcUfaUfuCfgAfuguUfgAfaUfuAfasUfsg AD-52763.1 A-108537.2aAfaCfcAfuAfuUfuguAfgUfuCfuCfcsCfsa AD-52764.1 A-108553.2aAfuUfaGfaUfuGfcuuCfaCfuAfuGfgsAfsg AD-52765.1 A-108569.2aAfuAfuUfcAfaUfauaAfuGfuUfuGfusUfsg AD-52766.1 A-108585.2uUfuGfuUfuUfcCfgggAfuUfgCfaUfusGfsg AD-52767.1 A-108507.2uUfaAfuGfuCfcAfuggAfcUfaCfcUfgsAfsu AD-52768.1 A-108523.2aUfcCfaUfcUfaUfucgAfuGfuUfgAfasUfsu AD-52769.1 A-108539.2uAfuAfuCfuUfcUfcuaGfgCfcCfaAfcsCfsa AD-52770.1 A-108555.2uAfaUfuAfgAfuUfgcuUfcAfcUfaUfgsGfsa AD-52771.1 A-108571.2aAfgAfaUfaUfuCfaauAfuAfaUfgUfusUfsg AD-52772.1 A-108587.2aUfcUfuUfgUfuUfuccGfgGfaUfuGfcsAfsu AD-52773.1 A-108509.2aAfuUfaAfuGfuCfcauGfgAfcUfaCfcsUfsg AD-52774.1 A-108525.2uUfuGfuGfaUfcCfaucUfaUfuCfgAfusGfsu AD-52775.1 A-108541.2aUfgGfaGfuAfuAfucuUfcUfcUfaGfgsCfsc AD-52776.1 A-108557.2uUfgUfcUfuUfcCfaguCfuUfcCfaAfcsUfsc AD-52777.1 A-108573.2aAfaGfaAfuAfuUfcaaUfaUfaAfuGfusUfsu AD-52778.1 A-108589.2aAfuCfuUfuGfuUfuucCfgGfgAfuUfgsCfsa AD-52779.1 A-108605.2uUfgCfuUfuGfuGfaucCfcAfaGfuAfgsAfsa AD-52780.1 A-108621.2uAfuAfuUfuAfcCfauuUfaGfgUfuGfusUfsu AD-52781.1 A-108637.2uUfcUfgAfaUfcUfguuGfgAfuGfgAfusCfsa AD-52782.1 A-108653.2uCfuUfuUfaAfaUfuugCfcUfcAfgUfusCfsa AD-52783.1 A-108669.2aCfaGfaUfuUfuUfacaCfaUfaCfuCfusGfsu AD-52784.1 A-108591.2aAfaUfcUfuUfgUfuuuCfcGfgGfaUfusGfsc AD-52785.1 A-108607.2uUfuGfcUfuUfgUfgauCfcCfaAfgUfasGfsa AD-52786.1 A-108623.2uUfaUfaUfuUfaCfcauUfuAfgGfuUfgsUfsu AD-52787.1 A-108639.2aAfuUfuGfcCfuCfaguUfcAfuUfcAfasAfsg AD-52788.1 A-108655.2uCfcUfuUfuAfaAfuuuGfcCfuCfaGfusUfsc AD-52789.1 A-108671.2uAfcAfgAfuUfuUfuacAfcAfuAfcUfcsUfsg AD-52791.1 A-108609.2uUfuUfgCfuUfuGfugaUfcCfcAfaGfusAfsg AD-52792.1 A-108625.2uUfgGfuUfuGfuUfauaUfuUfaCfcAfusUfsu AD-52793.1 A-108641.2aAfaUfuUfgCfcUfcagUfuCfaUfuCfasAfsa AD-52794.1 A-108657.2uGfcCfuUfuUfaAfauuUfgCfcUfcAfgsUfsu AD-52795.1 A-108673.2uUfaCfaGfaUfuUfuuaCfaCfaUfaCfusCfsu AD-52796.1 A-108595.2aAfaCfaCfcAfaAfucuUfuGfuUfuUfcsCfsg AD-52797.1 A-108611.2uUfaGfgUfuGfuUfuucUfcCfaCfaCfusCfsa AD-52798.1 A-108627.2uAfuAfaCfcUfuCfcauUfuUfgAfgAfcsUfsu AD-52799.1 A-108643.2uAfaAfuUfuGfcCfucaGfuUfcAfuUfcsAfsa AD-52800.1 A-108659.2uUfgCfcUfuUfuAfaauUfuGfcCfuCfasGfsu AD-52801.1 A-108675.2aUfuAfcAfgAfuUfuuuAfcAfcAfuAfcsUfsc AD-52802.1 A-108597.2aAfaAfcAfcCfaAfaucUfuUfgUfuUfusCfsc AD-52803.1 A-108613.2uUfuAfgGfuUfgUfuuuCfuCfcAfcAfcsUfsc AD-52804.1 A-108629.2uUfaUfaGfaGfuAfuaaCfcUfuCfcAfusUfsu AD-52805.1 A-108645.2uUfaAfaUfuUfgCfcucAfgUfuCfaUfusCfsa AD-52806.1 A-108661.2aUfuGfcCfuUfuUfaaaUfuUfgCfcUfcsAfsg AD-52807.1 A-108677.2uAfuUfaCfaGfaUfuuuUfaCfaCfaUfasCfsu AD-52808.1 A-108599.2uAfgAfaAfaCfaCfcaaAfuCfuUfuGfusUfsu AD-52809.1 A-108615.2aUfuUfaGfgUfuGfuuuUfcUfcCfaCfasCfsu AD-52810.1 A-108631.2uUfuAfuAfgAfgUfauaAfcCfuUfcCfasUfsu AD-52811.1 A-108647.2uUfuAfaAfuUfuGfccuCfaGfuUfcAfusUfsc AD-52812.1 A-108663.2uAfuUfgCfcUfuUfuaaAfuUfuGfcCfusCfsa AD-52813.1 A-108601.2aAfgUfaGfaAfaAfcacCfaAfaUfcUfusUfsg AD-52814.1 A-108617.2uAfuUfuAfcCfaUfuuaGfgUfuGfuUfusUfsc AD-52815.1 A-108633.2uUfgGfuUfgAfuUfuuaUfaGfaGfuAfusAfsa AD-52816.1 A-108649.2uUfuUfaAfaUfuUfgccUfcAfgUfuCfasUfsu AD-52817.1 A-108665.2uUfaUfuGfcCfuUfuuaAfaUfuUfgCfcsUfsc AD-52818.1 A-108603.2uUfuGfuGfaUfcCfcaaGfuAfgAfaAfasCfsa AD-52819.1 A-108619.2aUfaUfuUfaCfcAfuuuAfgGfuUfgUfusUfsu AD-52820.1 A-108635.2uUfuGfgUfuGfaUfuuuAfuAfgAfgUfasUfsa AD-52821.1 A-108651.2uUfuUfuAfaAfuUfugcCfuCfaGfuUfcsAfsu AD-52822.1 A-108667.2aAfgAfuUfuUfuAfcacAfuAfcUfcUfgsUfsg

TABLE 11 Results of single dose screen using ANGPTL3 GalNac-conjugateddsRNA Modified siRNAs were tested by transfection in Hep3b cells and byfree- uptake in primary cynomolgus monkey (PCH) cells at theabove-stated doses. 500 nM 100 nM 10 nM PCH PCH PCH STDEV STDEV STDEVSTDEV STDEV 10 nM 0.1 nM Celsis Celsis Celsis 10 nM 0.1 nM 500 nM 100 nM10 nM DUPLEX ID (RNAimax) (RNAimax) (FU) (FU) (FU) (RNAimax) (RNAimax)(FU) (FU) (FU) AD1955/naïve FU 0.93 0.93 1.01 0.91 1.17 0.02 0.08 0.090.00 0.07 AD1955/naïve FU 1.02 1.09 1.07 1.07 0.92 0.06 0.04 0.02 0.000.03 AD1955/naïve FU 1.06 0.99 0.93 1.02 0.93 0.03 0.00 0.09 0.01 0.02AD1955/naïve FU 1.05 0.90 1.05 1.03 1.03 0.04 0.02 0.01 0.05 0.01AD1955/naïve FU 1.06 1.08 0.90 0.97 1.03 0.02 0.01 0.02 0.04 0.09AD1955/naïve FU 0.90 1.03 1.05 1.00 0.94 0.04 0.03 0.01 0.04 0.05AD-45165 (TTR) 0.91 0.98 1.06 0.98 0.96 0.05 0.01 0.05 0.00 0.00AD-52953.1 0.06 0.34 0.15 0.17 0.46 0.00 0.01 0.00 0.01 0.01 AD-52954.10.09 0.39 0.17 0.20 0.55 0.00 0.01 0.00 0.01 0.00 AD-52955.1 0.11 0.590.38 0.41 0.75 0.01 0.04 0.02 0.01 0.12 AD-52956.1 0.31 0.94 0.79 0.941.17 0.01 0.00 0.02 0.06 0.02 AD-52957.1 0.13 0.61 0.35 0.38 0.73 0.010.00 0.01 0.00 0.04 AD-52958.1 0.19 0.74 0.66 0.71 0.97 0.01 0.01 0.020.07 0.06 AD-52960.1 0.14 0.59 0.31 0.32 0.55 0.01 0.01 0.00 0.02 0.02AD-52961.1 0.05 0.66 0.27 0.24 0.49 0.00 0.00 0.00 0.02 0.02 AD-52962.10.83 0.89 1.03 1.02 1.26 0.02 0.05 0.07 0.07 0.07 AD-52963.1 0.07 0.720.46 0.56 0.91 0.00 0.00 0.00 0.00 0.06 AD-52964.1 0.13 0.73 0.41 0.470.68 0.01 0.03 0.02 0.03 0.01 AD-52965.1 0.07 0.44 0.16 0.18 0.43 0.000.01 0.00 0.01 0.01 AD-52966.1 0.12 0.76 0.67 0.72 0.96 0.00 0.02 0.050.01 0.01 AD-52967.1 0.10 0.75 0.44 0.58 0.89 0.01 0.04 0.02 0.03 0.04AD-52968.1 1.01 0.96 0.87 0.91 1.15 0.00 0.01 0.09 0.03 0.02 AD-52969.10.04 0.46 0.22 0.29 0.59 0.00 0.00 0.01 0.02 0.04 AD-52970.1 0.06 0.450.27 0.30 0.51 0.00 0.00 0.01 0.02 0.00 AD-52971.1 0.08 0.55 0.20 0.220.45 0.00 0.00 0.01 0.02 0.05 AD-52972.1 0.10 0.73 0.41 0.49 0.81 0.000.01 0.01 0.02 0.01 AD-52973.1 0.11 0.73 0.36 0.46 0.75 0.01 0.01 0.030.02 0.02 AD-52974.1 1.00 0.95 1.00 1.09 1.27 0.01 0.01 0.08 0.05 0.06AD-52975.1 0.07 0.54 0.25 0.34 0.66 0.00 0.01 0.01 0.01 0.03 AD-52976.10.17 0.59 0.35 0.41 0.65 0.00 0.02 0.04 0.01 0.01 AD-52977.1 0.07 0.450.16 0.25 0.50 0.01 0.02 0.00 0.02 0.03 AD-52978.1 0.10 0.72 0.39 0.530.77 0.00 0.02 0.00 0.08 0.03 AD-52979.1 0.54 0.92 0.99 1.12 1.28 0.010.02 0.02 0.04 0.05 AD-52980.1 0.29 0.85 0.67 0.85 1.03 0.01 0.01 0.050.05 0.04 AD-52981.1 0.07 0.44 0.20 0.26 0.59 0.01 0.02 0.00 0.00 0.03AD-52982.1 0.28 0.87 0.67 0.99 1.14 0.01 0.01 0.04 0.00 0.01 AD-52983.10.06 0.40 0.14 0.40 0.46 0.00 0.00 0.01 0.05 0.02 AD-52984.1 0.29 0.870.66 0.74 1.09 0.01 0.02 0.01 0.00 0.00 AD-52985.1 0.72 0.87 0.89 1.181.22 0.03 0.00 0.05 0.03 0.16 AD-52986.1 0.08 0.47 0.24 0.30 0.48 0.000.02 0.02 0.00 0.06 AD-52987.1 0.16 0.83 0.42 0.73 1.09 0.00 0.00 0.010.02 0.02 AD-52988.1 0.11 0.73 0.42 0.60 0.96 0.01 0.04 0.00 0.00 0.10AD-52989.1 0.05 0.48 0.15 0.42 0.46 0.00 0.02 0.00 0.02 0.00 AD-52990.10.14 0.86 0.33 0.45 0.77 0.00 0.01 0.00 0.02 0.05 AD-52991.1 0.16 0.860.58 0.69 1.05 0.00 0.00 0.02 0.00 0.02 AD-52992.1 0.08 0.65 0.42 0.560.90 0.00 0.01 0.02 0.01 0.00 AD-52993.1 0.13 0.87 0.53 0.76 1.08 0.020.03 0.04 0.04 0.00 AD-52994.1 0.10 0.52 0.28 0.33 0.53 0.01 0.00 0.020.00 0.01 AD-52995.1 0.06 0.56 0.19 0.41 0.60 0.00 0.01 0.04 0.02 0.05AD-52996.1 0.09 0.68 0.26 0.47 0.68 0.00 0.03 0.01 0.04 0.01 AD-52997.10.59 1.03 0.87 0.51 1.25 0.05 0.01 0.00 0.01 0.01 AD-52998.1 0.09 0.790.44 0.55 0.85 0.00 0.00 0.04 0.03 0.10 AD-52999.1 0.08 0.57 0.17 0.360.84 0.01 0.00 0.01 0.02 0.00 AD-53000.1 0.38 0.94 0.58 0.67 0.85 0.010.02 0.03 0.03 0.02 AD-53001.1 0.05 0.48 0.21 0.18 0.40 0.00 0.00 0.010.00 0.05 AD-53002.1 0.07 0.65 0.43 0.48 0.80 0.00 0.05 0.04 0.01 0.02AD-53003.1 0.05 0.46 0.31 0.34 0.56 0.01 0.01 0.00 0.02 0.05 AD-53004.10.05 0.36 0.29 0.66 0.57 0.00 0.01 0.03 0.35 0.02 AD-53005.1 0.05 0.720.32 0.58 0.83 0.01 0.00 0.01 0.29 0.00 AD-53006.1 0.21 0.82 0.66 0.771.03 0.01 0.00 0.02 0.07 0.02 AD-53007.1 0.12 0.76 0.55 0.73 0.74 0.010.00 0.00 0.08 0.20 AD-53008.1 0.07 0.68 0.28 0.36 0.84 0.00 0.02 0.010.05 0.03 AD-53009.1 0.10 0.61 0.48 0.60 0.91 0.00 0.02 0.01 0.01 0.06AD-53010.1 0.05 0.58 0.47 0.54 0.84 0.00 0.02 0.00 0.02 0.03 AD-53011.10.07 0.65 0.29 0.34 0.84 0.00 0.03 0.07 0.01 0.04 AD-53012.1 0.06 0.550.36 0.45 0.70 0.00 0.03 0.02 0.02 0.00 AD-53013.1 0.11 0.85 0.59 0.701.01 0.00 0.00 0.03 0.03 0.02 AD-53014.1 0.16 0.78 0.61 0.78 1.11 0.000.02 0.01 0.05 0.00 AD-53015.1 0.03 0.35 0.25 0.37 0.46 0.01 0.01 0.010.00 0.01 AD-53016.1 0.03 0.56 0.40 0.58 1.01 0.00 0.01 0.02 0.06 0.09AD-53017.1 0.07 0.71 0.64 0.78 0.98 0.00 0.01 0.01 0.05 0.00 AD-53018.10.30 0.96 0.75 0.97 1.14 0.00 0.02 0.02 0.03 0.05 AD-53019.1 0.27 0.990.77 1.05 1.31 0.00 0.01 0.01 0.04 0.00 AD-53020.1 0.04 0.64 0.32 0.450.69 0.00 0.00 0.03 0.02 0.03 AD-53021.1 0.04 0.68 0.36 0.48 0.70 0.010.01 0.02 0.07 0.00 AD-53022.1 0.05 0.76 0.36 0.59 1.04 0.01 0.01 0.020.03 0.06 AD-53023.1 0.10 0.83 0.69 0.84 0.97 0.01 0.01 0.06 0.02 0.01AD-53024.1 0.09 0.44 0.23 0.23 0.44 0.00 0.00 0.03 0.01 0.02 AD-53025.10.09 0.87 0.58 0.80 1.09 0.00 0.03 0.01 0.04 0.04 AD-53026.1 0.05 0.600.35 0.46 0.77 0.01 0.01 0.02 0.05 0.03 AD-53027.1 0.02 0.32 0.26 0.300.45 0.00 0.01 0.02 0.03 0.02 AD-53028.1 0.19 0.82 0.77 0.95 1.04 0.010.04 0.05 0.01 0.03 AD-53029.1 0.02 0.52 0.32 0.41 0.72 0.00 0.00 0.010.02 0.07 AD-53030.1 0.09 0.42 0.15 0.16 0.46 0.00 0.00 0.00 0.00 0.02AD-53031.1 0.12 0.79 0.63 0.73 1.04 0.02 0.05 0.02 0.04 0.03 AD-53032.10.12 0.71 0.41 0.59 0.90 0.01 0.00 0.02 0.04 0.00 AD-53033.1 0.02 0.480.20 0.21 0.51 0.00 0.02 0.02 0.01 0.00 AD-53034.1 0.04 0.52 0.31 0.360.71 0.00 0.01 0.07 0.02 0.01 AD-53035.1 0.02 0.63 0.34 0.50 0.85 0.000.02 0.03 0.00 0.03 AD-53036.1 0.10 0.57 0.31 0.35 0.65 0.01 0.01 0.030.03 0.01 AD-53037.1 0.08 0.47 0.27 0.36 0.60 0.00 0.02 0.01 0.03 0.01AD-53038.1 0.05 0.85 0.48 0.63 1.08 0.00 0.05 0.00 0.02 0.05 AD-53039.10.08 0.82 0.45 0.64 0.97 0.00 0.01 0.01 0.03 0.00 AD-53040.1 0.05 0.790.46 0.62 0.97 0.01 0.01 0.01 0.05 0.06 AD-53041.1 0.06 0.72 0.59 0.610.86 0.00 0.01 0.05 0.06 0.03 AD-53042.1 0.08 0.85 0.30 0.35 0.81 0.010.00 0.00 0.03 0.03 AD-53043.1 0.63 1.00 0.92 1.04 1.07 0.03 0.00 0.060.03 0.07 AD-53044.1 0.05 0.91 0.35 0.61 0.97 0.01 0.01 0.01 0.04 0.02AD-53045.1 0.20 1.00 0.85 1.00 0.98 0.00 0.03 0.04 0.01 0.04 AD-53046.10.07 0.70 0.44 0.62 1.12 0.00 0.01 0.03 0.00 0.09 AD-53059.1 0.35 1.040.75 0.85 0.86 0.01 0.01 0.03 0.02 0.04 AD-53060.1 0.34 0.85 0.72 0.960.82 0.00 0.01 0.02 0.01 0.02 AD-53061.1 0.17 0.94 0.36 0.37 0.59 0.000.00 0.02 0.00 0.02 AD-53062.1 0.09 0.76 0.43 0.47 0.69 0.01 0.01 0.010.03 0.01 AD-53063.1 0.06 0.48 0.18 0.16 0.25 0.00 0.01 0.01 0.01 0.02AD-53064.1 0.07 0.59 0.22 0.22 0.48 0.01 0.02 0.01 0.02 0.06 AD-53065.10.08 0.97 0.45 0.39 0.64 0.01 0.01 0.02 0.01 0.01 AD-53066.1 0.12 0.990.73 0.67 0.88 0.01 0.03 0.01 0.01 0.05 AD-53067.1 0.12 1.08 0.59 0.600.79 0.00 0.12 0.01 0.01 0.03 AD-53068.1 0.09 0.98 0.46 0.59 0.83 0.000.03 0.04 0.07 0.05 AD-53069.1 0.04 0.69 0.35 0.43 0.59 0.00 0.01 0.010.04 0.01 AD-53070.1 0.17 1.12 0.88 0.83 0.98 0.00 0.01 0.04 0.00 0.01AD-53071.1 0.07 0.70 0.23 0.23 0.43 0.00 0.00 0.02 0.00 0.01 AD-53072.10.10 0.90 0.49 0.48 0.75 0.01 0.05 0.00 0.01 0.02 AD-53073.1 0.07 0.630.27 0.30 0.43 0.00 0.00 0.01 0.01 0.00 AD-53074.1 0.07 0.88 0.46 0.490.62 0.01 0.08 0.01 0.06 0.03 AD-53075.1 0.05 0.76 0.29 0.35 0.50 0.010.01 0.00 0.02 0.03 AD-53076.1 0.09 0.80 0.31 0.40 0.54 0.01 0.01 0.020.05 0.02 AD-53077.1 0.07 0.96 0.29 0.28 0.49 0.00 0.03 0.00 0.01 0.01AD-53078.1 0.16 0.95 0.51 0.51 0.70 0.00 0.04 0.01 0.01 0.06 AD-53079.10.08 0.96 0.59 0.67 0.83 0.00 0.02 0.01 0.03 0.01 AD-53080.1 0.04 0.630.20 0.22 0.43 0.00 0.01 0.00 0.01 0.01 AD-53081.1 0.16 1.02 0.63 0.750.87 0.00 0.09 0.00 0.02 0.05 AD-53082.1 0.06 0.94 0.50 0.52 0.66 0.010.06 0.02 0.03 0.03 AD-53083.1 0.14 0.87 0.48 0.50 0.80 0.01 0.02 0.040.06 0.01 AD-53084.1 0.12 0.95 0.50 0.47 0.72 0.01 0.03 0.04 0.00 0.00AD-53085.1 0.27 1.02 0.68 0.81 0.99 0.01 0.01 0.01 0.05 0.02 AD-53086.10.05 0.60 0.26 0.25 0.48 0.00 0.01 0.03 0.00 0.01 AD-53087.1 0.05 0.560.32 0.39 0.53 0.00 0.01 0.01 0.03 0.02 AD-53088.1 0.09 0.89 0.53 0.690.87 0.00 0.01 0.02 0.04 0.02 AD-53089.1 0.29 0.97 0.58 0.57 0.78 0.010.00 0.02 0.02 0.02 AD-53090.1 0.13 0.86 0.56 0.55 0.73 0.00 0.01 0.010.03 0.00 AD-53091.1 0.12 0.82 0.27 0.35 0.66 0.00 0.03 0.03 0.01 0.07AD-53092.1 0.05 0.66 0.26 0.29 0.42 0.00 0.01 0.02 0.04 0.02 AD-53093.10.08 0.68 0.36 0.44 0.55 0.00 0.02 0.03 0.04 0.10 AD-53094.1 0.32 1.001.05 0.92 1.11 0.02 0.01 0.01 0.00 0.03 AD-53095.1 0.14 0.77 0.29 0.290.49 0.00 0.02 0.00 0.01 0.01 AD-53096.1 0.30 0.96 0.61 0.57 0.73 0.030.01 0.02 0.02 0.01 AD-53097.1 0.37 0.97 0.67 0.82 0.86 0.01 0.01 0.010.02 0.01 AD-53098.1 0.06 0.65 0.22 0.30 0.43 0.00 0.03 0.03 0.00 0.01AD-53099.1 0.34 0.99 0.61 0.81 0.91 0.00 0.00 0.04 0.02 0.06 AD-53100.10.31 1.04 0.95 1.03 1.00 0.02 0.01 0.06 0.02 0.17 AD-53101.1 0.46 0.930.63 0.69 0.78 0.00 0.01 0.04 0.03 0.04 AD-53102.1 0.23 0.80 0.60 0.550.66 0.00 0.03 0.01 0.02 0.03 AD-53103.1 0.05 0.61 0.27 0.32 0.50 0.010.02 0.00 0.01 0.00 AD-53104.1 0.13 0.80 0.64 0.68 0.77 0.00 0.02 0.030.01 0.05 AD-53105.1 0.15 0.77 0.43 0.65 0.77 0.01 0.03 0.02 0.02 0.05AD-53106.1 0.16 0.87 0.72 0.70 0.83 0.01 0.02 0.00 0.00 0.04 AD-53107.10.19 0.95 0.62 0.65 0.90 0.00 0.02 0.01 0.03 0.04 AD-53108.1 0.22 0.940.60 0.68 0.81 0.00 0.01 0.00 0.03 0.04 AD-53109.1 0.16 1.01 0.82 0.780.96 0.01 0.08 0.04 0.01 0.07 AD-53110.1 0.10 0.86 0.79 0.77 0.94 0.000.05 0.03 0.01 0.05 AD-53111.1 0.22 0.78 0.94 0.85 1.04 0.01 0.01 0.010.01 0.07 AD-53112.1 0.09 0.96 0.64 0.65 0.86 0.01 0.02 0.07 0.07 0.00AD-53113.1 0.10 0.97 0.71 0.77 0.88 0.01 0.05 0.01 0.02 0.01 AD-53114.10.19 0.83 0.48 0.52 0.66 0.01 0.01 0.02 0.01 0.00 AD-53115.1 0.10 0.590.42 0.44 0.66 0.01 0.03 0.04 0.00 0.02 AD-53116.1 0.11 0.87 0.82 0.850.95 0.00 0.05 0.05 0.05 0.05 AD-53117.1 0.52 0.64 1.21 1.00 1.08 0.010.03 0.09 0.04 0.07 AD-53118.1 0.19 1.04 0.60 0.72 0.94 0.00 0.07 0.020.05 0.06 AD-53119.1 0.06 0.77 0.44 0.47 0.64 0.01 0.03 0.00 0.01 0.01AD-53120.1 0.10 0.97 0.78 0.89 1.01 0.01 0.04 0.05 0.01 0.04 AD-53121.10.23 0.80 0.58 0.69 0.90 0.01 0.02 0.04 0.02 0.06 AD-53122.1 0.09 0.800.90 0.94 1.09 0.01 0.07 0.02 0.04 0.10 AD-53123.1 0.27 0.74 0.95 0.930.97 0.00 0.01 0.03 0.01 0.08 AD-53124.1 0.08 0.81 0.33 0.34 0.61 0.010.02 0.00 0.01 0.01 AD-53125.1 0.08 0.82 0.34 0.38 0.58 0.00 0.02 0.000.01 0.07 AD-53126.1 0.15 0.95 0.70 0.86 1.06 0.01 0.04 0.05 0.02 0.00AD-53127.1 0.21 0.81 0.62 0.75 0.91 0.02 0.04 0.01 0.03 0.00 AD-53128.10.08 0.79 0.80 1.14 1.09 0.00 0.06 0.04 0.01 0.03 AD-53129.1 0.48 0.781.05 1.00 1.10 0.00 0.01 0.06 0.01 0.03 AD-53130.1 0.25 1.08 0.63 0.720.88 0.01 0.02 0.00 0.01 0.00 AD-53131.1 0.14 0.96 0.54 0.57 0.81 0.020.02 0.05 0.01 0.04 AD-53132.1 0.03 0.54 0.24 0.27 0.49 0.00 0.02 0.020.00 0.01 AD-53133.1 0.12 0.76 0.50 0.67 0.93 0.00 0.03 0.01 0.01 0.06AD-53134.1 0.28 0.86 1.14 0.81 0.97 0.01 0.04 0.05 0.02 0.04 AD-53135.10.47 0.74 1.03 0.94 1.09 0.01 0.03 0.04 0.07 0.04 AD-53136.1 0.09 0.990.64 0.69 0.94 0.01 0.05 0.01 0.05 0.02 AD-53137.1 0.08 0.75 0.39 0.390.59 0.01 0.03 0.00 0.00 0.00 AD-53138.1 0.04 0.71 0.33 0.34 0.60 0.000.02 0.00 0.03 0.00 AD-53139.1 0.11 0.76 0.55 0.66 0.84 0.01 0.01 0.060.01 0.02 AD-53140.1 0.09 0.71 0.64 0.71 0.86 0.00 0.04 0.01 0.02 0.02AD-53141.1 0.24 1.09 0.77 0.91 0.93 0.00 0.01 0.00 0.06 0.00 AD-53142.10.13 0.95 0.55 0.70 0.82 0.01 0.03 0.03 0.04 0.02 AD-53143.1 0.13 0.910.67 0.83 0.94 0.01 0.00 0.03 0.03 0.07 AD-53144.1 0.10 0.72 0.54 0.690.84 0.01 0.03 0.01 0.03 0.00 AD-53145.1 0.08 0.72 0.70 0.78 0.88 0.010.03 0.01 0.08 0.02 AD-53146.1 0.83 1.07 0.85 0.96 0.98 0.01 0.06 0.000.05 0.00 AD-53147.1 0.08 0.56 0.27 0.34 0.47 0.00 0.01 0.01 0.01 0.01AD-53148.1 0.06 0.81 0.61 0.68 0.74 0.01 0.00 0.03 0.06 0.05 AD-53149.10.23 0.86 0.71 0.83 0.92 0.01 0.02 0.06 0.02 0.03 AD-53150.1 0.41 0.701.03 1.09 1.03 0.03 0.06 0.03 0.04 0.01

TABLE 12 Dose response screen results for ANGPTL3 GalNac-conjugateddsRNA sequences A subset of active siRNAs from the single dose screen(refer to data in Table 11) was tested in a dose response experiment byfree uptake in PCH cells. A subset of these active siRNAs was alsotested in dose response in Hep3B cells by transfection. IC₅₀ (nM) Freeuptake Transfection (RNAiMax) AD-53063.1 1.60 0.03 AD-53001.1 2.27 0.01AD-53015.1 2.90 0.02 AD-52953.1 2.94 0.03 AD-52986.1 3.30 0.03AD-53024.1 3.42 0.02 AD-53033.1 3.42 0.02 AD-53027.1 3.84 0.01AD-53030.1 3.90 0.03 AD-53080.1 4.08 0.04 AD-53073.1 4.20 0.05AD-52965.1 4.63 ND AD-53092.1 5.37 ND AD-53132.1 5.54 ND AD-52983.1 5.55ND AD-52954.1 5.67 ND AD-52961.1 6.37 ND AD-52994.1 6.43 ND AD-53098.16.58 ND AD-52970.1 6.71 ND AD-53075.1 6.74 ND AD-53086.1 7.08 NDAD-52971.1 7.50 ND AD-53064.1 8.33 ND AD-53147.1 8.34 ND AD-52969.1 8.86ND AD-53077.1 8.98 ND AD-52981.1 9.44 ND AD-52977.1 10.45 ND AD-53071.111.19 ND AD-52960.1 13.03 ND AD-53095.1 21.31 ND AD-53103.1 21.92 ND

TABLE 13 Results of single dose screen using sequences listed in Table10. 10 0.1 0.025 STDEV STDEV STDEV Duplex nM nM nM 10 nM 0.1 nM 0.025 nMAD-52719.1 0.01 0.60 0.35 0.000 0.093 0.002 AD-52717.1 0.02 0.31 0.320.001 0.014 0.008 AD-52713.1 0.02 0.37 0.36 0.001 0.011 0.007 AD-52711.10.03 0.22 0.23 0.005 0.011 0.009 AD-52718.1 0.03 0.31 0.39 0.000 0.0250.023 AD-52687.1 0.03 0.37 0.38 0.005 0.020 0.002 AD-52699.1 0.03 0.250.21 0.002 0.011 0.002 AD-52679.1 0.03 0.51 0.24 0.345 0.008 AD-52689.10.03 0.44 0.42 0.000 0.039 0.002 AD-52700.1 0.03 0.56 0.57 0.005 0.0440.020 AD-52637.1 0.04 0.27 0.23 0.001 0.003 0.005 AD-52730.1 0.04 0.610.59 0.005 0.053 0.014 AD-52725.1 0.04 0.62 0.61 0.002 0.027 0.012AD-52688.1 0.04 0.23 0.20 0.006 0.012 0.011 AD-52661.1 0.04 0.61 0.250.001 0.449 0.009 AD-52667.1 0.04 0.28 0.22 0.004 0.018 0.013 AD-52665.10.04 0.43 0.48 0.007 0.019 0.009 AD-52638.1 0.04 0.28 0.25 0.000 0.0160.027 AD-52724.1 0.05 0.86 0.76 0.001 0.055 0.011 AD-52705.1 0.05 0.740.65 0.004 0.022 0.016 AD-52708.1 0.05 0.53 0.52 0.001 0.034 0.013AD-52659.1 0.05 0.56 0.48 0.000 0.000 0.033 AD-52678.1 0.05 0.53 0.530.002 0.034 0.000 AD-52670.1 0.05 0.35 0.33 0.002 0.009 0.003 AD-52695.10.05 0.63 0.67 0.001 0.012 0.013 AD-52704.1 0.05 0.55 0.53 0.002 0.0050.034 AD-52683.1 0.05 0.36 0.28 0.002 0.021 0.011 AD-52673.1 0.05 0.220.19 0.023 0.010 0.002 AD-52721.1 0.05 0.60 0.53 0.003 0.006 0.029AD-52710.1 0.05 0.56 0.40 0.007 0.073 0.000 AD-52714.1 0.05 0.40 0.510.000 0.016 0.003 AD-52686.1 0.05 0.57 0.60 0.003 0.014 0.000 AD-52645.10.05 0.62 0.59 0.004 0.030 0.003 AD-52662.1 0.05 0.55 0.52 0.002 0.0300.008 AD-52720.1 0.05 0.50 0.46 0.003 0.007 0.011 AD-52654.1 0.05 0.290.36 0.008 0.037 0.014 AD-52680.1 0.06 0.48 0.41 0.001 0.019 0.026AD-52723.1 0.06 0.84 0.76 0.001 0.041 0.004 AD-52726.1 0.06 0.72 0.660.003 0.028 0.016 AD-52701.1 0.06 0.67 0.39 0.001 0.003 0.002 AD-52694.10.06 0.68 0.59 0.004 0.040 0.012 AD-52685.1 0.06 0.30 0.25 0.002 0.0130.016 AD-52728.1 0.06 0.80 0.79 0.005 0.043 0.015 AD-52676.1 0.06 0.680.67 0.002 0.023 0.029 AD-52639.1 0.06 0.47 0.45 0.000 0.005 0.007AD-52722.1 0.06 0.81 0.93 0.005 0.004 0.027 AD-52682.1 0.06 0.87 0.730.009 0.038 0.014 AD-52660.1 0.07 0.69 0.68 0.002 0.014 0.017 AD-52709.10.07 0.89 0.82 0.001 0.013 0.020 AD-52643.1 0.07 0.27 0.24 0.006 0.0160.012 AD-52696.1 0.07 0.53 0.46 0.003 0.026 0.007 AD-52657.1 0.08 0.600.58 0.008 0.030 0.006 AD-52706.1 0.08 0.84 0.78 0.001 0.021 0.019AD-52653.1 0.08 0.41 0.45 0.057 0.004 0.029 AD-52656.1 0.08 0.65 0.500.004 0.022 0.012 AD-52693.1 0.09 0.61 0.62 0.007 0.021 0.018 AD-52692.10.09 0.54 0.52 0.023 0.018 0.033 AD-52674.1 0.10 0.79 0.64 0.001 0.0080.028 AD-52648.1 0.10 0.67 0.53 0.002 0.013 0.028 AD-52651.1 0.10 0.840.73 0.000 0.000 0.007 AD-52641.1 0.10 0.62 0.50 0.004 0.172 0.002AD-52707.1 0.10 0.92 0.81 0.001 0.018 0.032 AD-52671.1 0.11 0.87 0.840.005 0.034 0.025 AD-52650.1 0.12 0.88 0.94 0.007 0.013 0.041 AD-52642.10.12 0.90 0.76 0.015 0.022 0.004 AD-52675.1 0.13 0.94 0.89 0.001 0.0180.044 AD-52647.1 0.13 0.80 0.79 0.031 0.008 0.023 AD-52716.1 0.14 0.610.69 0.010 0.060 0.013 AD-52649.1 0.14 0.31 0.29 0.136 0.020 0.006AD-52677.1 0.16 1.01 0.72 0.059 0.040 0.007 AD-52697.1 0.16 0.86 0.770.012 0.021 0.015 AD-52715.1 0.17 0.90 0.89 0.005 0.009 0.022 AD-52691.10.18 0.93 0.88 0.004 0.036 0.017 AD-52698.1 0.20 0.97 0.87 0.010 0.0280.000 AD-52672.1 0.20 0.70 0.66 0.170 0.014 0.019 AD-52712.1 0.29 0.920.90 0.007 0.036 0.004 AD-52690.1 0.30 0.95 0.85 0.115 0.032 0.004AD-52640.1 0.30 1.04 0.91 0.018 0.046 0.013 AD-52684.1 0.31 0.90 0.940.014 0.018 0.014 AD-52666.1 0.32 1.04 0.91 0.013 0.005 0.004 AD-52703.10.32 1.02 0.96 0.016 0.015 0.005 AD-52729.1 0.33 1.02 0.87 0.032 0.0200.008 AD-52668.1 0.35 0.94 0.90 0.029 0.046 0.026 AD-52681.1 0.57 1.000.99 0.003 0.034 0.039 AD-52702.1 0.72 1.02 0.92 0.658 0.060 0.014AD-52727.1 0.73 1.03 0.91 0.004 0.065 0.027 AD-52663.1 0.78 1.05 0.960.027 0.010 0.005 AD-52669.1 0.91 0.91 0.94 0.004 0.049 0.032 AD-19550.95 0.84 0.95 0.005 0.021 0.019 AD-1955 0.97 1.07 1.03 0.000 0.0210.015 AD-1955 1.01 1.08 1.01 0.035 0.011 0.005 mock 1.02 0.96 0.97 0.0300.037 0.005 AD-1955 1.08 1.03 1.02 0.032 0.051 0.005 AD-52652.1 1.131.11 1.02 0.028 0.043 0.020 AD-52658.1 1.33 1.10 0.93 0.091 0.043 0.018AD-52664.1 1.49 0.95 0.88 0.438 0.019 0.009 AD-52752.1 0.03 0.43 0.690.002 0.015 0.017 AD-52741.1 0.03 0.56 0.86 0.001 0.044 0.021 AD-52804.10.03 0.49 0.89 0.001 0.002 0.017 AD-52764.1 0.03 0.54 0.79 0.005 0.0160.078 AD-52770.1 0.03 0.58 0.78 0.000 0.006 0.027 AD-52735.1 0.03 0.310.46 0.003 0.031 0.009 AD-52810.1 0.03 0.67 0.86 0.001 0.013 0.025AD-52759.1 0.03 0.54 0.79 0.000 0.018 0.023 AD-52736.1 0.03 0.51 0.600.004 0.012 0.023 AD-52775.1 0.03 0.54 0.73 0.005 0.024 0.022 AD-52758.10.03 0.57 0.78 0.001 0.014 0.050 AD-52743.1 0.03 0.45 0.67 0.002 0.0180.033 AD-52747.1 0.04 0.57 0.84 0.002 0.061 0.058 AD-52819.1 0.04 0.260.45 0.005 0.001 0.022 AD-52765.1 0.04 0.68 0.83 0.000 0.013 0.053AD-52754.1 0.04 0.76 1.00 0.000 0.007 0.015 AD-52787.1 0.05 0.55 0.680.001 0.043 0.060 AD-52791.1 0.05 0.70 0.91 0.001 0.014 0.084 AD-52811.10.05 0.73 0.84 0.002 0.014 0.058 AD-52817.1 0.05 0.77 0.92 0.003 0.0110.031 AD-52745.1 0.06 0.62 0.77 0.007 0.021 0.000 AD-52749.1 0.06 0.630.88 0.005 0.037 0.043 AD-52740.1 0.06 0.83 0.94 0.007 0.012 0.051AD-52796.1 0.06 0.72 0.92 0.003 0.021 0.054 AD-52820.1 0.06 0.90 0.870.001 0.026 0.064 AD-52809.1 0.06 0.76 0.90 0.001 0.037 0.027 AD-52760.10.06 0.81 0.97 0.001 0.056 0.047 AD-52767.1 0.07 0.55 0.55 0.001 0.0160.013 AD-52734.1 0.07 0.61 0.64 0.004 0.003 0.003 AD-52794.1 0.07 0.940.87 0.007 0.014 0.051 AD-52797.1 0.07 0.69 0.87 0.004 0.000 0.038AD-52737.1 0.08 0.70 0.84 0.004 0.031 0.012 AD-52812.1 0.08 0.75 0.880.004 0.000 0.056 AD-52748.1 0.08 0.70 0.89 0.001 0.010 0.009 AD-52782.10.08 0.68 0.78 0.004 0.023 0.011 AD-52816.1 0.08 0.71 0.88 0.003 0.0420.060 AD-52763.1 0.08 0.68 0.77 0.002 0.013 0.026 AD-52788.1 0.08 0.891.00 0.004 0.017 0.034 AD-52762.1 0.08 0.78 0.91 0.007 0.046 0.009AD-52785.1 0.08 0.88 0.95 0.002 0.004 0.019 AD-52800.1 0.09 0.82 0.940.001 0.040 0.005 AD-52792.1 0.09 0.93 0.94 0.002 0.018 0.037 AD-52784.10.10 0.84 0.92 0.000 0.066 0.032 AD-52746.1 0.10 0.82 0.93 0.002 0.0600.059 AD-52814.1 0.10 0.85 0.88 0.002 0.042 0.013 AD-52751.1 0.10 0.880.98 0.005 0.030 0.067 AD-52786.1 0.10 0.81 0.81 0.006 0.028 0.048AD-52755.1 0.10 0.93 0.99 0.003 0.032 0.048 AD-52808.1 0.11 0.98 0.920.000 0.038 0.032 AD-52815.1 0.11 0.96 0.96 0.002 0.009 0.000 AD-52805.10.11 0.79 0.86 0.003 0.050 0.008 AD-52777.1 0.11 0.88 0.94 0.001 0.0650.000 AD-52756.1 0.11 0.92 0.91 0.003 0.032 0.004 AD-52733.1 0.12 0.660.65 0.005 0.071 0.022 AD-52739.1 0.13 0.83 0.95 0.002 0.008 0.061AD-52780.1 0.13 0.70 0.67 0.012 0.021 0.059 AD-52798.1 0.13 0.64 0.970.001 0.006 0.038 AD-52776.1 0.14 0.97 0.94 0.011 0.029 0.023 AD-52753.10.15 0.88 1.09 0.001 0.048 0.005 AD-52778.1 0.16 0.76 0.69 0.003 0.0670.003 AD-52744.1 0.16 0.90 0.91 0.002 0.000 0.049 AD-52750.1 0.16 0.871.01 0.000 0.060 0.055 AD-52774.1 0.17 0.71 0.89 0.002 0.010 0.017AD-52803.1 0.18 0.87 0.92 0.015 0.026 0.040 AD-52821.1 0.18 0.86 0.870.005 0.046 0.055 AD-52781.1 0.18 0.78 0.66 0.008 0.000 0.023 AD-52779.10.20 0.83 0.66 0.002 0.024 0.016 AD-52793.1 0.20 0.74 0.88 0.010 0.0250.069 AD-52799.1 0.20 0.75 1.01 0.005 0.018 0.010 AD-52761.1 0.22 0.830.92 0.000 0.024 0.023 AD-52768.1 0.22 0.96 0.97 0.001 ND 0.028AD-52757.1 0.23 1.02 0.95 0.018 0.040 0.042 AD-52806.1 0.24 0.96 0.870.011 0.084 0.055 AD-52771.1 0.25 0.92 0.98 0.010 0.018 0.048 AD-52802.10.30 0.95 1.00 0.010 0.019 0.005 AD-52731.1 0.30 0.85 0.75 0.001 0.0670.022 AD-52813.1 0.30 1.07 0.98 0.001 0.109 0.014 AD-52742.1 0.31 0.951.03 0.005 0.028 0.056 AD-52766.1 0.35 0.97 1.00 0.010 0.024 0.044AD-52732.1 0.41 0.79 0.73 0.004 0.016 0.039 AD-52773.1 0.43 0.99 0.920.004 0.029 0.022 AD-52772.1 0.43 1.00 1.02 0.006 0.000 0.065 AD-52822.10.44 0.68 0.81 0.004 0.010 0.016 AD-52783.1 0.45 0.66 0.76 0.009 0.0360.019 AD-52789.1 0.50 0.68 0.78 0.010 0.053 0.004 AD-52795.1 0.50 0.820.69 0.000 0.080 0.054 AD-52801.1 0.54 0.70 0.79 0.018 0.038 0.035AD-52807.1 0.57 0.76 0.93 0.006 0.011 0.032 AD-52769.1 0.76 0.97 0.920.015 0.085 0.045 AD-1955 0.90 0.96 1.04 0.018 0.165 0.010 AD-52818.10.92 1.03 0.92 0.009 0.010 0.063 AD-1955 1.01 0.90 0.96 0.005 0.0310.019 AD-1955 1.05 1.09 1.00 0.046 0.085 0.005 AD-1955 1.05 1.07 1.000.010 0.031 0.039 mock 1.20 0.98 0.92 0.000 0.014 0.005 mock 1.25 0.991.00 0.006 0.005 0.034

TABLE 14 Results of a dose response screen using a subset of sequencesfrom Table 13. A subset of active ANGPTL3 siRNAs from Table 10 weretested by transfection in Hep3B cells in dose response screens. DuplexIC50 (nM) AD-52819.1 0.0036 AD-52667.1 0.0037 AD-52638.1 0.0048AD-52673.1 0.0049 AD-52711.1 0.0050 AD-52661.1 0.0054 AD-52654.1 0.0058AD-52637.1 0.0058 AD-52643.1 0.0060 AD-52685.1 0.0062 AD-52670.1 0.0064AD-52679.1 0.0064 AD-52649.1 0.0066 AD-52683.1 0.0069 AD-52688.1 0.0071AD-52717.1 0.0072 AD-52699.1 0.0073 AD-52714.1 0.0086 AD-52718.1 0.0088AD-52735.1 0.0093 AD-52653.1 0.0102 AD-52687.1 0.0109 AD-52680.1 0.0120AD-52713.1 0.0133 AD-52720.1 0.0143 AD-52639.1 0.0161 AD-52696.1 0.0163AD-52662.1 0.0179 AD-52659.1 0.0180 AD-52710.1 0.0195 AD-52689.1 0.0216AD-52787.1 0.0242 AD-52765.1 0.0318

TABLE 15 IDs of duplex pairs for which both an unconjuaged and aGalNac-conjugated version were synthesized and tested These duplexeshave the same sequence and modification pattern. Unconjugated duplex IDGalNac conjugated duplex ID AD-52637.1 AD-52953.1 AD-52638.1 AD-52954.1AD-52639.1 AD-52955.1 AD-52640.1 AD-52956.1 AD-52641.1 AD-52957.1AD-52642.1 AD-52958.1 AD-52643.1 None None AD-52960.1 None AD-52961.1AD-52645.1 AD-52962.1 AD-52647.1 AD-52963.1 AD-52648.1 AD-52964.1AD-52649.1 AD-52965.1 AD-52650.1 AD-52966.1 AD-52651.1 AD-52967.1AD-52652.1 AD-52968.1 AD-52653.1 AD-52969.1 AD-52654.1 AD-52970.1 NoneAD-52971.1 AD-52656.1 AD-52972.1 AD-52657.1 AD-52973.1 AD-52658.1AD-52974.1 AD-52659.1 AD-52975.1 AD-52660.1 AD-52976.1 AD-52661.1AD-52977.1 AD-52662.1 AD-52978.1 AD-52663.1 AD-52979.1 AD-52664.1AD-52980.1 AD-52665.1 AD-52981.1 AD-52666.1 AD-52982.1 AD-52667.1AD-52983.1 AD-52668.1 AD-52984.1 AD-52669.1 AD-52985.1 AD-52670.1AD-52986.1 AD-52671.1 AD-52987.1 AD-52672.1 AD-52988.1 AD-52673.1AD-52989.1 AD-52674.1 AD-52990.1 AD-52675.1 AD-52991.1 AD-52676.1AD-52992.1 AD-52677.1 AD-52993.1 AD-52678.1 AD-52994.1 AD-52679.1AD-52995.1 AD-52680.1 AD-52996.1 AD-52681.1 AD-52997.1 AD-52682.1AD-52998.1 AD-52683.1 AD-52999.1 AD-52684.1 AD-53000.1 AD-52685.1AD-53001.1 AD-52686.1 AD-53002.1 AD-52687.1 AD-53003.1 AD-52688.1AD-53004.1 AD-52689.1 AD-53005.1 AD-52690.1 AD-53006.1 AD-52691.1AD-53007.1 AD-52692.1 AD-53008.1 AD-52693.1 AD-53009.1 AD-52694.1AD-53010.1 AD-52695.1 AD-53011.1 AD-52696.1 AD-53012.1 AD-52697.1AD-53013.1 AD-52698.1 AD-53014.1 AD-52699.1 AD-53015.1 AD-52700.1AD-53016.1 AD-52701.1 AD-53017.1 AD-52702.1 AD-53018.1 AD-52703.1AD-53019.1 AD-52704.1 AD-53020.1 AD-52705.1 AD-53021.1 AD-52706.1AD-53022.1 AD-52707.1 AD-53023.1 AD-52708.1 AD-53024.1 AD-52709.1AD-53025.1 AD-52710.1 AD-53026.1 AD-52711.1 AD-53027.1 AD-52712.1AD-53028.1 AD-52713.1 AD-53029.1 AD-52714.1 AD-53030.1 AD-52715.1AD-53031.1 AD-52716.1 AD-53032.1 AD-52717.1 AD-53033.1 AD-52718.1AD-53034.1 AD-52719.1 AD-53035.1 AD-52720.1 AD-53036.1 AD-52721.1AD-53037.1 AD-52722.1 AD-53038.1 AD-52723.1 AD-53039.1 AD-52724.1AD-53040.1 AD-52725.1 AD-53041.1 AD-52726.1 AD-53042.1 AD-52727.1AD-53043.1 AD-52728.1 AD-53044.1 AD-52729.1 AD-53045.1 AD-52730.1AD-53046.1 AD-52731.1 AD-53059.1 AD-52732.1 AD-53060.1 AD-52733.1AD-53061.1 AD-52734.1 AD-53062.1 AD-52735.1 AD-53063.1 AD-52736.1AD-53064.1 AD-52737.1 AD-53065.1 None AD-53066.1 AD-52739.1 AD-53067.1AD-52740.1 AD-53068.1 AD-52741.1 AD-53069.1 AD-52742.1 AD-53070.1AD-52743.1 AD-53071.1 AD-52744.1 AD-53072.1 AD-52745.1 AD-53073.1AD-52746.1 AD-53074.1 AD-52747.1 AD-53075.1 AD-52748.1 AD-53076.1AD-52749.1 AD-53077.1 AD-52750.1 AD-53078.1 AD-52751.1 AD-53079.1AD-52752.1 AD-53080.1 AD-52753.1 AD-53081.1 AD-52754.1 AD-53082.1AD-52755.1 AD-53083.1 AD-52756.1 AD-53084.1 AD-52757.1 AD-53085.1AD-52758.1 AD-53086.1 AD-52759.1 AD-53087.1 AD-52760.1 AD-53088.1AD-52761.1 AD-53089.1 AD-52762.1 AD-53090.1 AD-52763.1 AD-53091.1AD-52764.1 AD-53092.1 AD-52765.1 AD-53093.1 AD-52766.1 AD-53094.1AD-52767.1 AD-53095.1 AD-52768.1 AD-53096.1 AD-52769.1 AD-53097.1AD-52770.1 AD-53098.1 AD-52771.1 AD-53099.1 AD-52772.1 AD-53100.1AD-52773.1 AD-53101.1 AD-52774.1 AD-53102.1 AD-52775.1 AD-53103.1AD-52776.1 AD-53104.1 AD-52777.1 AD-53105.1 AD-52778.1 AD-53106.1AD-52779.1 AD-53107.1 AD-52780.1 AD-53108.1 AD-52781.1 AD-53109.1AD-52782.1 AD-53110.1 AD-52783.1 AD-53111.1 AD-52784.1 AD-53112.1AD-52785.1 AD-53113.1 AD-52786.1 AD-53114.1 AD-52787.1 AD-53115.1AD-52788.1 AD-53116.1 AD-52789.1 AD-53117.1 None AD-53118.1 AD-52791.1AD-53119.1 AD-52792.1 AD-53120.1 AD-52793.1 AD-53121.1 AD-52794.1AD-53122.1 AD-52795.1 AD-53123.1 AD-52796.1 AD-53124.1 AD-52797.1AD-53125.1 AD-52798.1 AD-53126.1 AD-52799.1 AD-53127.1 AD-52800.1AD-53128.1 AD-52801.1 AD-53129.1 AD-52802.1 AD-53130.1 AD-52803.1AD-53131.1 AD-52804.1 AD-53132.1 AD-52805.1 AD-53133.1 AD-52806.1AD-53134.1 AD-52807.1 AD-53135.1 AD-52808.1 AD-53136.1 AD-52809.1AD-53137.1 AD-52810.1 AD-53138.1 AD-52811.1 AD-53139.1 AD-52812.1AD-53140.1 AD-52813.1 AD-53141.1 AD-52814.1 AD-53142.1 AD-52815.1AD-53143.1 AD-52816.1 AD-53144.1 AD-52817.1 AD-53145.1 AD-52818.1AD-53146.1 AD-52819.1 AD-53147.1 AD-52820.1 AD-53148.1 AD-52821.1AD-53149.1 AD-52822.1 AD-53150.1In Vivo Tests

Example 3

Test Articles

In vivo experiments were conducted using dsRNA sequences of theinvention. The dsRNA sequence used in the experiments wasGalNac-conjugated AD-52981 (“ANG”, sense sequence:AfcAfuAfuUfuGfAfUfcAfgUfcUfuUfuUfL96 (SEQ ID NO: 657); antisensesequence: aAfaAfaGfaCfuGfaucAfaAfuAfuGfusUfsg (SEQ ID NO: 842)). ThedsRNA sequence used as a negative control was luciferase-conjugatedAD-48399B1 (“Luc”, sense sequence: CfaCfuUfaCfgCfuGfaGfuAfcUfuCfgAfL96(SEQ ID NO: 1728), antisense sequence:uCfgAfaGfuAfcUfcAfgCfgUfaAfgUfgsAfsu (SEQ ID NO: 1729)). Also used as anegative control was GalNal-conjugated AD-1955 containing alternating2′-methyl and 2′ fluoro modifications.

Experimental Procedure

The dsRNA sequences were tested in C57BL/6 (WT) and ob/ob mice. WT micereceived five daily doses of dsRNAs in PBS, Luc at 20 mg/kg, or ANG at 5or 20 mg/kg; and ob/ob mice received five daily doses of NPLs formulatedwith Luc at 20 mg/kg or ANG at 20 mg/kg. All test articles wereadministered by subcutaneous injection according to the procedure shownin FIG. 1 . Specifically, five daily doses of the test articles wereadministered on five consecutive days (day 0, 1, 2, 3 and 4), and bloodsamples were collected 5, 3 or 1 day prior to administration, as well ason days 0, 1, 2, 3, 4, 7, 9, 11, 15, 18, 21, 25, 30, 37, 45 and 50post-administration. The collected blood samples were used to measurethe expression of ANGPTL3 protein using an ELISA assay. Levels of serumtriglycerides (TGs), low density lipoprotein cholesterol (LDLc), highdensity lipoprotein cholesterol (HDLc) and total cholesterol (TC) werealso measured using an Olympus Analyzer.

Results

Shown in FIG. 2 , Panel A, are levels of murine ANGPTL3 (mANGPTL3,protein measured in WT mice after administration of control or ANG at 5or 20 mg/kg. Also shown in FIG. 2 , Panel B are levels of mANGPTL3protein measured in ob/ob mice after administration of control or ANG at20 mg/kg. The data indicates that, for both WT and ob/ob mice,administration of ANG results in decreased levels of mANGPTL3 protein,as compared to controls.

Shown in FIG. 3 , Panel A, are levels of LDL-c measured in WT mice afteradministration of control or ANG at 20 mg/kg. Shown in FIG. 3 , Panel Bare levels of LDL-c measured in ob/ob mice after administration ofcontrol or ANG at 20 mg/kg. The data indicates that administration ofANG causes decreased levels of LDL-c, particularly in ob/ob mice, ascompared to controls.

Shown in FIG. 4 , Panel A, are levels of triglycerides measured in WTmice after administration of control or ANG at 20 mg/kg. Shown in FIG. 4, Panel B are levels of triglycerides measured in ob/ob mice afteradministration of control or ANG at 20 mg/kg. The data indicates thatadministration of ANG causes decreased levels of tryglycerides,particularly, in ob/ob mice, as compared to controls.

Shown in FIG. 5 , Panel A and B are levels of total cholesterol (TC)measured in WT and ob/ob mice, respectively, after administration ofcontrol or ANG at 20 mg/kg. The data indicates that administration ofANG causes a moderate decrease in TC levels in ob/ob mice, but not in WTmice. Similarly, administration of ANG causes a moderate decrease inHDL-c levels in ob/ob mice, but not in WT mice, as is shown in thegraphs in FIG. 6 .

Example 4

Test Article

The effect of a single injection of dsRNA sequence of the invention onthe level of ANGPTL3 protein was tested. The dsRNA sequence used in theexperiments was GalNac-conjugated AD-52981 (“ANG”, sense sequence:AfcAfuAfuUfuGfAfUfcAfgUfcUfuUfuUfL96 (SEQ ID NO: 657); antisensesequence: aAfaAfaGfaCfuGfaucAfaAfuAfuGfusUfsg (SEQ ID NO: 842)). PBS wasused as a negative control.

Experimental Procedure

The dsRNA sequences were tested in Human PCS Transgenic mousecharacterized by liver-specific expression of full-length human PCSK9gene. Human PCS transgenic mice were dosed with the AD-52981 or PBSusing a single subcutaneous injection. The mice were divided into fourgroups, each group consisting of two males and two females. Each groupreceived an injection of PBS or a 5 mg/kg, 20 mg/kg or 60 mg/kg dose ofAD-52981. Blood samples were collected at day 1 and day 0 prior todosing, and at 72 hours post dosing. ANGPTL3 protein levels weremeasured by ELISA and compared to levels at day 1 and day 0 prior todosing.

Results

Shown in FIG. 7 , are levels of murine ANGPTL3 protein (mANGPTL3)measured in Human PCS transgenic mice. The data shown is expressedrelative to PBS control and represents an average for 2 males and 2females in each group. Error bars represent standard deviation. The dataindicates that administration of a single injection of AD-52981 reducesthe levels of ANGPTL3 protein in the mice in a dose-dependent manner,with the dose of 60 mg/kg decreasing the levels of ANGPTL3 protein morethan five-fold (see FIG. 7 ).

SEQUENCES

>gi|41327750|ref|NM_014495.21| Homo sapiens angiopoietin-like 3(ANGPTL3), mRNA SEQ ID NO: 1TTCCAGAAGAAAACAGTTCCACGTTGCTTGAAATTGAAAATCAAGATAAAAATGTTCACAATTAAGCTCCTTCTTTTTATTGTTCCTCTAGTTATTTCCTCCAGAATTGATCAAGACAATTCATCATTTGATTCTCTATCTCCAGAGCCAAAATCAAGATTTGCTATGTTAGACGATGTAAAAATTTTAGCCAATGGCCTCCTTCAGTTGGGACATGGTCTTAAAGACTTTGTCCATAAGACGAAGGGCCAAATTAATGACATATTTCAAAAACTCAACATATTTGATCAGTCTTTTTATGATCTATCGCTGCAAACCAGTGAAATCAAAGAAGAAGAAAAGGAACTGAGAAGAACTACATATAAACTACAAGTCAAAAATGAAGAGGTAAAGAATATGTCACTTGAACTCAACTCAAAACTTGAAAGCCTCCTAGAAGAAAAAATTCTACTTCAACAAAAAGTGAAATATTTAGAAGAGCAACTAACTAACTTAATTCAAAATCAACCTGAAACTCCAGAACACCCAGAAGTAACTTCACTTAAAACTTTTGTAGAAAAACAAGATAATAGCATCAAAGACCTTCTCCAGACCGTGGAAGACCAATATAAACAATTAAACCAACAGCATAGTCAAATAAAAGAAATAGAAAATCAGCTCAGAAGGACTAGTATTCAAGAACCCACAGAAATTTCTCTATCTTCCAAGCCAAGAGCACCAAGAACTACTCCCTTTCTTCAGTTGAATGAAATAAGAAATGTAAAACATGATGGCATTCCTGCTGAATGTACCACCATTTATAACAGAGGTGAACATACAAGTGGCATGTATGCCATCAGACCCAGCAACTCTCAAGTTTTTCATGTCTACTGTGATGTTATATCAGGTAGTCCATGGACATTAATTCAACATCGAATAGATGGATCACAAAACTTCAATGAAACGTGGGAGAACTACAAATATGGTTTTGGGAGGCTTGATGGAGAATTTTGGTTGGGCCTAGAGAAGATATACTCCATAGTGAAGCAATCTAATTATGTTTTACGAATTGAGTTGGAAGACTGGAAAGACAACAAACATTATATTGAATATTCTTTTTACTTGGGAAATCACGAAACCAACTATACGCTACATCTAGTTGCGATTACTGGCAATGTCCCCAATGCAATCCCGGAAAACAAAGATTTGGTGTTTTCTACTTGGGATCACAAAGCAAAAGGACACTTCAACTGTCCAGAGGGTTATTCAGGAGGCTGGTGGTGGCATGATGAGTGTGGAGAAAACAACCTAAATGGTAAATATAACAAACCAAGAGCAAAATCTAAGCCAGAGAGGAGAAGAGGATTATCTTGGAAGTCTCAAAATGGAAGGTTATACTCTATAAAATCAACCAAAATGTTGATCCATCCAACAGATTCAGAAAGCTTTGAATGAACTGAGGCAAATTTAAAAGGCAATAATTTAAACATTAACCTCATTCCAAGTTAATGTGGTCTAATAATCTGGTATTAAATCCTTAAGAGAAAGCTTGAGAAATAGATTTTTTTTATCTTAAAGTCACTGTCTATTTAAGATTAAACATACAATCACATAACCTTAAAGAATACCGTTTACATTTCTCAATCAAAATTCTTATAATACTATTTGTTTTAAATTTTGTGATGTGGGAATCAATTTTAGATGGTCACAATCTAGATTATAATCAATAGGTGAACTTATTAAATAACTTTTCTAAATAAAAAATTTAGAGACTTTTATTTTAAAAGGCATCATATGAGCTAATATCACAACTTTCCCAGTTTAAAAAACTAGTACTCTTGTTAAAACTCTAAACTTGACTAAATACAGAGGACTGGTAATTGTACAGTTCTTAAATGTTGTAGTATTAATTTCAAAACTAAAAATCGTCAGCACAGAGTATGTGTAAAAATCTGTAATACAAATTTTTAAACTGATGCTTCATTTTGCTACAAAATAATTTGGAGTAAATGTTTGATATGATTTATTTATGAAACCTAATGAAGCAGAATTAAATACTGTATTAAAATAAGTTCGCTGTCTTTAAACAAATGGAGATGACTACTAAGTCACATTGACTTTAACATGAGGTATCACTATACCTTATT>gi|297278846|ref|XM_001086114.2| PREDICTED: Macaca mulatta angiopoietin-like3 (ANGPTL3), mRNA SEQ ID NO: 2ATATATAGAGTTAAGAAGTCTAGGTCTGCTTCCAGAAGAACACAGTTCCACGTTGCTTGAAATTGAAAATCAGGATAAAAATGTTCACAATTAAGCTCCTTCTTTTTATTGTTCCTCTAGTTATTTCCTCCAGAATTGACCAAGACAATTCATCATTTGATTCTGTATCTCCAGAGCCAAAATCAAGATTTGCTATGTTAGACGATGTAAAAATTTTAGCCAATGGCCTCCTTCAGTTGGGACATGGTCTTAAAGACTTTGTCCATAAGACTAAGGGCCAAATTAATGACATATTTCAAAAACTCAACATATTTGATCAGTCTTTTTATGATCTATCACTGCAAACCAGTGAAATCAAAGAAGAAGAAAAGGAACTGAGAAGAACTACATATAAACTACAAGTCAAAAATGAAGAGGTAAAGAATATGTCACTTGAACTCAACTCAAAACTTGAAAGCCTCCTAGAAGAAAAAATTCTACTTCAACAAAAAGTGAAATATTTAGAAGAGCAACTAACTAACTTAATTCAAAATCAACCTGAAACTCCAGAACATCCAGAAGTAACTTCACTTAAAAGTTTTGTAGAAAAACAAGATAATAGCATCAAAGACCTTCTCCAGACTGTGGAAGAACAATATAAGCAATTAAACCAACAGCACAGTCAAATAAAAGAAATAGAAAATCAGCTCAGAATGACTAATATTCAAGAACCCACAGAAATTTCTCTATCTTCCAAGCCAAGAGCACCAAGAACTACTCCCTTTCTTCAGCTGAATGAAATAAGAAATGTAAAACATGATGGCATTCCTGCTGATTGTACCACCATTTACAATAGAGGTGAACATATAAGTGGCATGTATGCCATCAGACCCAGCAACTCTCAAGTTTTTCATGTCTACTGTGATGTTGTATCAGGTAAAACCTGTCTAAGGAGAATAGATGGATCACAAAACTTCAATGAAACGTGGGAGAACTACAAATATGGTTTCGGGAGGCTTGATGGAGAATTCTGGTTGGGCCTAGAGAAGATATACTCCATAGTGAAGCAATCTAATTACGTTTTACGAATTGAGTTGGAAGACTGGAAAGACAACAAACATTATATTGAATATTCTTTTTACTTGGGAAATCACGAAACCAACTATACGCTACATGTAGTTAAGATTACTGGCAATGTCCCCAATGCAATCCCGGAAAACAAAGATTTGGTGTTTTCTACTTGGGATCACAAAGCAAAAGGACACTTCAGCTGTCCAGAGAGTTATTCAGGAGGCTGGTGGTGGCATGATGAGTGTGGAGAAAACAACCTAAATGGTAAATATAACAAACCAAGAACAAAATCTAAGCCAGAGCGGAGAAGAGGATTATCCTGGAAGTCTCAAAATGGAAGGTTATACTCTATAAAATCAACCAAAATGTTGATCCATCCAACAGATTCAGAAAGCTTTGAATGAACTGAGGCAAATTTAAAAGGCAATAAATTAAACATTAAACTCATTCCAAGTTAATGTGGTTTAATAATCTGGTATTAAATCCTTAAGAGAAGGCTTGAGAAATAGATTTTTTTATCTTAAAGTCACTGTCAATTTAAGATTAAACATACAATCACATAACCTTAAAGAATACCATTTACATTTCTCAATCAAAATTCCTACAACACTATTTGTTTTATATTTTGTGATGTGGGAATCAATTTTAGATGGTCGCAATCTAAATTATAATCAACAGGTGAACTTACTAAATAACTTTTCTAAATAAAAAACTTAGAGACTTTAATTTTAAAAGTCATCATATGAGCTAATATCACAATTTTCCCAGTTTAAAAAACTAGTTTTCTTGTTAAAACTCTAAACTTGACTAAATAAAGAGGACTGATAATTATACAGTTCTTAAATTTGTTGTAATATTAATTTCAAAACTAAAAATTGTCAGCACAGAGTATGTGTAAAAATCTGTAATATAAATTTTTAAACTGATGCCTCATTTTGCTACAAAATAATCTGGAGTAAATTTTTGATAGGATTTATTTATGAAACCTAATGAAGCAGGATTAAATACTGTATTAAAATAGGTTCGCTGTCTTTTAAACAAATGGAGATGATGATTACTAAGTCACATTGACTTTAATATGAGGTATCACTATACCTTA>gi|142388354|ref|NM_013913.3| Mus musculus angiopoietin-like 3(Angpt13), mRNA SEQ ID NO: 3CAGGAGGGAGAAGTTCCAAATTGCTTAAAATTGAATAATTGAGACAAAAAATGCACACAATTAAATTATTCCTTTTTGTTGTTCCTTTAGTAATTGCATCCAGAGTGGATCCAGACCTTTCATCATTTGATTCTGCACCTTCAGAGCCAAAATCAAGATTTGCTATGTTGGATGATGTCAAAATTTTAGCGAATGGCCTCCTGCAGCTGGGTCATGGACTTAAAGATTTTGTCCATAAGACTAAGGGACAAATTAACGACATATTTCAGAAGCTCAACATATTTGATCAGTCTTTTTATGACCTATCACTTCGAACCAATGAAATCAAAGAAGAGGAAAAGGAGCTAAGAAGAACTACATCTACACTACAAGTTAAAAACGAGGAGGTGAAGAACATGTCAGTAGAACTGAACTCAAAGCTTGAGAGTCTGCTGGAAGAGAAGACAGCCCTTCAACACAAGGTCAGGGCTTTGGAGGAGCAGCTAACCAACTTAATTCTAAGCCCAGCTGGGGCTCAGGAGCACCCAGAAGTAACATCACTCAAAAGTTTTGTAGAACAGCAAGACAACAGCATAAGAGAACTCCTCCAGAGTGTGGAAGAACAGTATAAACAATTAAGTCAACAGCACATGCAGATAAAAGAAATAGAAAAGCAGCTCAGAAAGACTGGTATTCAAGAACCCTCAGAAAATTCTCTTTCTTCTAAATCAAGAGCACCAAGAACTACTCCCCCTCTTCAACTGAACGAAACAGAAAATACAGAACAAGATGACCTTCCTGCCGACTGCTCTGCCGTTTATAACAGAGGCGAACATACAAGTGGCGTGTACACTATTAAACCAAGAAACTCCCAAGGGTTTAATGTCTACTGTGATACCCAATCAGGCAGTCCATGGACATTAATTCAACACCGGAAAGATGGCTCACAGGACTTCAACGAAACATGGGAAAACTACGAAAAGGGCTTTGGGAGGCTCGATGGAGAATTTTGGTTGGGCCTAGAGAAGATCTATGCTATAGTCCAACAGTCTAACTACATTTTACGACTCGAGCTACAAGACTGGAAAGACAGCAAGCACTACGTTGAATACTCCTTTCACCTGGGCAGTCACGAAACCAACTACACGCTACATGTGGCTGAGATTGCTGGCAATATCCCTGGGGCCCTCCCAGAGCACACAGACCTGATGTTTTCTACATGGAATCACAGAGCAAAGGGACAGCTCTACTGTCCAGAAAGTTACTCAGGTGGCTGGTGGTGGAATGACATATGTGGAGAAAACAACCTAAATGGAAAATACAACAAACCCAGAACCAAATCCAGACCAGAGAGAAGAAGAGGGATCTACTGGAGACCTCAGAGCAGAAAGCTCTATGCTATCAAATCATCCAAAATGATGCTCCAGCCCACCACCTAAGAAGCTTCAACTGAACTGAGACAAAATAAAAGATCAATAAATTAAATATTAAAGTCCTCCCGATCACTGTAGTAATCTGGTATTAAAATTTTAATGGAAAGCTTGAGAATTGAATTTCAATTAGGTTTAAACTCATTGTTAAGATCAGATATCACCGAATCAACGTAAACAAAATTTATC>gi|68163568|ref|NM_001025065.1| Rattus norvegicus angiopoietin-like 3(Angpt13), mRNA SEQ ID NO: 4GACGTTCCAAATTGCTTGAAATTGAATAATTGAAACAAAAATGCACACAATTAAGCTGCTCCTTTTTGTTGTTCCTCTAGTAATTTCGTCCAGAGTTGATCCAGACCTTTCGCCATTTGATTCTGTACCGTCAGAGCCAAAATCAAGATTTGCTATGTTGGATGATGTCAAAATTTTAGCCAATGGCCTCCTGCAGCTGGGTCATGGTCTTAAAGATTTTGTCCATAAGACAAAGGGACAAATTAATGACATATTTCAGAAGCTCAACATATTTGATCAGTGTTTTTATGACCTATCACTTCAAACCAATGAAATCAAAGAAGAGGAAAAGGAGCTAAGAAGAACCACATCTAAACTACAAGTTAAAAACGAAGAGGTGAAGAATATGTCACTTGAACTGAACTCAAAGCTTGAAAGTCTACTGGAGGAGAAGATGGCGCTCCAACACAGAGTCAGGGCTTTGGAGGAACAGCTGACCAGCTTGGTTCAGAACCCGCCTGGGGCTCGGGAGCACCCAGAGGTAACGTCACTTAAAAGTTTTGTAGAACAGCAAGATAACAGCATAAGAGAACTCCTCCAGAGTGTGGAAGAACAATATAAACAACTAAGTCAACAGCACATTCAGATAAAAGAAATAGAAAATCAGCTCAGAAAGACTGGCATTCAAGAACCCACTGAAAATTCTCTTTATTCTAAACCAAGAGCACCAAGAACTACTCCCCCTCTTCATCTGAAGGAAGCAAAAAATATAGAACAAGATGATCTGCCTGCTGACTGCTCTGCCATTTATAACAGAGGTGAACATACAAGTGGCGTGTATACTATTAGACCAAGCAGCTCTCAAGTGTTTAATGTCTACTGTGACACCCAATCAGGCACTCCACGGACATTAATTCAACACCGGAAAGATGGCTCTCAAAACTTCAACCAAACGTGGGAAAACTACGAAAAGGGTTTTGGGAGGCTTGATGGTAAAGTGATTTCCTTGCATCACTCACTTATCTGTTGATTTAATAGTATTAGTTGGGTGTGTTGACACAGGCCTGAGACCATAGCGCTTTTGGGCAAGGGGGGAGGAGGAGCAGCAGGTGAATTGAAAGTTCAAGACCAGTCTGGGCCACACATTGATACTCCTTCTCGACATTAAGAATTATAAATTAAGCAGCAATTATAAAATGGGCTGTGGAAATGTAACAATAAGCAAAAGCAGACCCCAGTCTTCATAAAACTGATTGGTAAATATTATCCATGATAGCAACTGCAATGATCTCATTGTACTTATCACTACTGCATGCCTGCAGTATGCTTGTTGAAACTTAATTCTATAGTTCATGGTTATCATAAGTCTTATTAAGGAACATAGTATACGCCATTGGCTCTAGTGAGGGGCCATGCTACAAATGAGCTGCAAAGATAGCAGTATAGAGCTCTTTCAGTGATATCCTAAGCACAACGTAACACAGGTGAAATGGGCTGGAGGCACAGTTGTGGTGGAACACGCGGCCAGCAGGACACTGGGACTGATCCCCAGCAGCACAAAGAAAGTGATAGGAACACAGAGCGAGAGTTAGAAGGGACAGGGTCACCGTCAGAGATACGGTGTCTAACTCCTGCAACCCTACCTGTAATTATTCCATATTATAAACATATACTATATAACTGTGGGTCTCTGCATGTTCTAGAATATGAATTCTTAATCAAAAA Reverse Complement of SEQ ID NO: 1 SEQ ID NO: 5AATAAGGTATAGTGATACCTCATGTTAAAGTCAATGTGACTTAGTAGTCATCTCCATTTGTTTAAAGACAGCGAACTTATTTTAATACAGTATTTAATTCTGCTTCATTAGGTTTCATAAATAAATCATATCAAACATTTACTCCAAATTATTTTGTAGCAAAATGAAGCATCAGTTTAAAAATTTGTATTACAGATTTTTACACATACTCTGTGCTGACGATTTTTAGTTTTGAAATTAATACTACAACATTTAAGAACTGTACAATTACCAGTCCTCTGTATTTAGTCAAGTTTAGAGTTTTAACAAGAGTACTAGTTTTTTAAACTGGGAAAGTTGTGATATTAGCTCATATGATGCCTTTTAAAATAAAAGTCTCTAAATTTTTTATTTAGAAAAGTTATTTAATAAGTTCACCTATTGATTATAATCTAGATTGTGACCATCTAAAATTGATTCCCACATCACAAAATTTAAAACAAATAGTATTATAAGAATTTTGATTGAGAAATGTAAACGGTATTCTTTAAGGTTATGTGATTGTATGTTTAATCTTAAATAGACAGTGACTTTAAGATAAAAAAAATCTATTTCTCAAGCTTTCTCTTAAGGATTTAATACCAGATTATTAGACCACATTAACTTGGAATGAGGTTAATGTTTAAATTATTGCCTTTTAAATTTGCCTCAGTTCATTCAAAGCTTTCTGAATCTGTTGGATGGATCAACATTTTGGTTGATTTTATAGAGTATAACCTTCCATTTTGAGACTTCCAAGATAATCCTCTTCTCCTCTCTGGCTTAGATTTTGCTCTTGGTTTGTTATATTTACCATTTAGGTTGTTTTCTCCACACTCATCATGCCACCACCAGCCTCCTGAATAACCCTCTGGACAGTTGAAGTGTCCTTTTGCTTTGTGATCCCAAGTAGAAAACACCAAATCTTTGTTTTCCGGGATTGCATTGGGGACATTGCCAGTAATCGCAACTAGATGTAGCGTATAGTTGGTTTCGTGATTTCCCAAGTAAAAAGAATATTCAATATAATGTTTGTTGTCTTTCCAGTCTTCCAACTCAATTCGTAAAACATAATTAGATTGCTTCACTATGGAGTATATCTTCTCTAGGCCCAACCAAAATTCTCCATCAAGCCTCCCAAAACCATATTTGTAGTTCTCCCACGTTTCATTGAAGTTTTGTGATCCATCTATTCGATGTTGAATTAATGTCCATGGACTACCTGATATAACATCACAGTAGACATGAAAAACTTGAGAGTTGCTGGGTCTGATGGCATACATGCCACTTGTATGTTCACCTCTGTTATAAATGGTGGTACATTCAGCAGGAATGCCATCATGTTTTACATTTCTTATTTCATTCAACTGAAGAAAGGGAGTAGTTCTTGGTGCTCTTGGCTTGGAAGATAGAGAAATTTCTGTGGGTTCTTGAATACTAGTCCTTCTGAGCTGATTTTCTATTTCTTTTATTTGACTATGCTGTTGGTTTAATTGTTTATATTGGTCTTCCACGGTCTGGAGAAGGTCTTTGATGCTATTATCTTGTTTTTCTACAAAAGTTTTAAGTGAAGTTACTTCTGGGTGTTCTGGAGTTTCAGGTTGATTTTGAATTAAGTTAGTTAGTTGCTCTTCTAAATATTTCACTTTTTGTTGAAGTAGAATTTTTTCTTCTAGGAGGCTTTCAAGTTTTGAGTTGAGTTCAAGTGACATATTCTTTACCTCTTCATTTTTGACTTGTAGTTTATATGTAGTTCTTCTCAGTTCCTTTTCTTCTTCTTTGATTTCACTGGTTTGCAGCGATAGATCATAAAAAGACTGATCAAATATGTTGAGTTTTTGAAATATGTCATTAATTTGGCCCTTCGTCTTATGGACAAAGTCTTTAAGACCATGTCCCAACTGAAGGAGGCCATTGGCTAAAATTTTTACATCGTCTAACATAGCAAATCTTGATTTTGGCTCTGGAGATAGAGAATCAAATGATGAATTGTCTTGATCAATTCTGGAGGAAATAACTAGAGGAACAATAAAAAGAAGGAGCTTAATTGTGAACATTTTTATCTTGATTTTCAATTTCAAGCAACGTGGAACTGTTTTCTTCTGGAAReverse Complement of SEQ ID NO: 2 SEQ ID NO: 6TAAGGTATAGTGATACCTCATATTAAAGTCAATGTGACTTAGTAATCATCATCTCCATTTGTTTAAAAGACAGCGAACCTATTTTAATACAGTATTTAATCCTGCTTCATTAGGTTTCATAAATAAATCCTATCAAAAATTTACTCCAGATTATTTTGTAGCAAAATGAGGCATCAGTTTAAAAATTTATATTACAGATTTTTACACATACTCTGTGCTGACAATTTTTAGTTTTGAAATTAATATTACAACAAATTTAAGAACTGTATAATTATCAGTCCTCTTTATTTAGTCAAGTTTAGAGTTTTAACAAGAAAACTAGTTTTTTAAACTGGGAAAATTGTGATATTAGCTCATATGATGACTTTTAAAATTAAAGTCTCTAAGTTTTTTATTTAGAAAAGTTATTTAGTAAGTTCACCTGTTGATTATAATTTAGATTGCGACCATCTAAAATTGATTCCCACATCACAAAATATAAAACAAATAGTGTTGTAGGAATTTTGATTGAGAAATGTAAATGGTATTCTTTAAGGTTATGTGATTGTATGTTTAATCTTAAATTGACAGTGACTTTAAGATAAAAAAATCTATTTCTCAAGCCTTCTCTTAAGGATTTAATACCAGATTATTAAACCACATTAACTTGGAATGAGTTTAATGTTTAATTTATTGCCTTTTAAATTTGCCTCAGTTCATTCAAAGCTTTCTGAATCTGTTGGATGGATCAACATTTTGGTTGATTTTATAGAGTATAACCTTCCATTTTGAGACTTCCAGGATAATCCTCTTCTCCGCTCTGGCTTAGATTTTGTTCTTGGTTTGTTATATTTACCATTTAGGTTGTTTTCTCCACACTCATCATGCCACCACCAGCCTCCTGAATAACTCTCTGGACAGCTGAAGTGTCCTTTTGCTTTGTGATCCCAAGTAGAAAACACCAAATCTTTGTTTTCCGGGATTGCATTGGGGACATTGCCAGTAATCTTAACTACATGTAGCGTATAGTTGGTTTCGTGATTTCCCAAGTAAAAAGAATATTCAATATAATGTTTGTTGTCTTTCCAGTCTTCCAACTCAATTCGTAAAACGTAATTAGATTGCTTCACTATGGAGTATATCTTCTCTAGGCCCAACCAGAATTCTCCATCAAGCCTCCCGAAACCATATTTGTAGTTCTCCCACGTTTCATTGAAGTTTTGTGATCCATCTATTCTCCTTAGACAGGTTTTACCTGATACAACATCACAGTAGACATGAAAAACTTGAGAGTTGCTGGGTCTGATGGCATACATGCCACTTATATGTTCACCTCTATTGTAAATGGTGGTACAATCAGCAGGAATGCCATCATGTTTTACATTTCTTATTTCATTCAGCTGAAGAAAGGGAGTAGTTCTTGGTGCTCTTGGCTTGGAAGATAGAGAAATTTCTGTGGGTTCTTGAATATTAGTCATTCTGAGCTGATTTTCTATTTCTTTTATTTGACTGTGCTGTTGGTTTAATTGCTTATATTGTTCTTCCACAGTCTGGAGAAGGTCTTTGATGCTATTATCTTGTTTTTCTACAAAACTTTTAAGTGAAGTTACTTCTGGATGTTCTGGAGTTTCAGGTTGATTTTGAATTAAGTTAGTTAGTTGCTCTTCTAAATATTTCACTTTTTGTTGAAGTAGAATTTTTTCTTCTAGGAGGCTTTCAAGTTTTGAGTTGAGTTCAAGTGACATATTCTTTACCTCTTCATTTTTGACTTGTAGTTTATATGTAGTTCTTCTCAGTTCCTTTTCTTCTTCTTTGATTTCACTGGTTTGCAGTGATAGATCATAAAAAGACTGATCAAATATGTTGAGTTTTTGAAATATGTCATTAATTTGGCCCTTAGTCTTATGGACAAAGTCTTTAAGACCATGTCCCAACTGAAGGAGGCCATTGGCTAAAATTTTTACATCGTCTAACATAGCAAATCTTGATTTTGGCTCTGGAGATACAGAATCAAATGATGAATTGTCTTGGTCAATTCTGGAGGAAATAACTAGAGGAACAATAAAAAGAAGGAGCTTAATTGTGAACATTTTTATCCTGATTTTCAATTTCAAGCAACGTGGAACTGTGTTCTTCTGGAAGCAGACCTAGACTTCTTAACTCTATATATReverse Complement of SEQ ID NO: 3 SEQ ID NO: 7CAGGAGGGAGAAGTTCCAAATTGCTTAAAATTGAATAATTGAGACAAAAAATGCACACAATTAAATTATTCCTTTTTGTTGTTCCTTTAGTAATTGCATCCAGAGTGGATCCAGACCTTTCATCATTTGATTCTGCACCTTCAGAGCCAAAATCAAGATTTGCTATGTTGGATGATGTCAAAATTTTAGCGAATGGCCTCCTGCAGCTGGGTCATGGACTTAAAGATTTTGTCCATAAGACTAAGGGACAAATTAACGACATATTTCAGAAGCTCAACATATTTGATCAGTCTTTTTATGACCTATCACTTCGAACCAATGAAATCAAAGAAGAGGAAAAGGAGCTAAGAAGAACTACATCTACACTACAAGTTAAAAACGAGGAGGTGAAGAACATGTCAGTAGAACTGAACTCAAAGCTTGAGAGTCTGCTGGAAGAGAAGACAGCCCTTCAACACAAGGTCAGGGCTTTGGAGGAGCAGCTAACCAACTTAATTCTAAGCCCAGCTGGGGCTCAGGAGCACCCAGAAGTAACATCACTCAAAAGTTTTGTAGAACAGCAAGACAACAGCATAAGAGAACTCCTCCAGAGTGTGGAAGAACAGTATAAACAATTAAGTCAACAGCACATGCAGATAAAAGAAATAGAAAAGCAGCTCAGAAAGACTGGTATTCAAGAACCCTCAGAAAATTCTCTTTCTTCTAAATCAAGAGCACCAAGAACTACTCCCCCTCTTCAACTGAACGAAACAGAAAATACAGAACAAGATGACCTTCCTGCCGACTGCTCTGCCGTTTATAACAGAGGCGAACATACAAGTGGCGTGTACACTATTAAACCAAGAAACTCCCAAGGGTTTAATGTCTACTGTGATACCCAATCAGGCAGTCCATGGACATTAATTCAACACCGGAAAGATGGCTCACAGGACTTCAACGAAACATGGGAAAACTACGAAAAGGGCTTTGGGAGGCTCGATGGAGAATTTTGGTTGGGCCTAGAGAAGATCTATGCTATAGTCCAACAGTCTAACTACATTTTACGACTCGAGCTACAAGACTGGAAAGACAGCAAGCACTACGTTGAATACTCCTTTCACCTGGGCAGTCACGAAACCAACTACACGCTACATGTGGCTGAGATTGCTGGCAATATCCCTGGGGCCCTCCCAGAGCACACAGACCTGATGTTTTCTACATGGAATCACAGAGCAAAGGGACAGCTCTACTGTCCAGAAAGTTACTCAGGTGGCTGGTGGTGGAATGACATATGTGGAGAAAACAACCTAAATGGAAAATACAACAAACCCAGAACCAAATCCAGACCAGAGAGAAGAAGAGGGATCTACTGGAGACCTCAGAGCAGAAAGCTCTATGCTATCAAATCATCCAAAATGATGCTCCAGCCCACCACCTAAGAAGCTTCAACTGAACTGAGACAAAATAAAAGATCAATAAATTAAATATTAAAGTCCTCCCGATCACTGTAGTAATCTGGTATTAAAATTTTAATGGAAAGCTTGAGAATTGAATTTCAATTAGGTTTAAACTCATTGTTAAGATCAGATATCACCGAATCAACGTAAACAAAATTTATCReverse Complement of SEQ ID NO: 4 SEQ ID NO: 8TTTTTTTTTTTTTTTTTTTTTTTTTGATTTTAAGTATCTGTTTATTTTTTATTTTTTTACTTATTTTTATAGTTTTGTTTTACAATCAAATAGAATTCATATTCTAGAACATGCAGAGACCCACAGTTATATAGTATATGTTTATAATATGGAATAATTACAGGTAGGGTTGCAGGAGTTAGACACCGTATCTCTGACGGTGACCCTGTCCCTTCTAACTCTCGCTCTGTGTTCCTATCACTTTCTTTGTGCTGCTGGGGATCAGTCCCAGTGTCCTGCTGGCCGCGTGTTCCACCACAACTGTGCCTCCAGCCCATTTCACCTGTGTTACGTTGTGCTTAGGATATCACTGAAAGAGCTCTATACTGCTATCTTTGCAGCTCATTTGTAGCATGGCCCCTCACTAGAGCCAATGGCGTATACTATGTTCCTTAATAAGACTTATGATAACCATGAACTATAGAATTAAGTTTCAACAAGCATACTGCAGGCATGCAGTAGTGATAAGTACAATGAGATCATTGCAGTTGCTATCATGGATAATATTTACCAATCAGTTTTATGAAGACTGGGGTCTGCTTTTGCTTATTGTTACATTTCCACAGCCCATTTTATAATTGCTGCTTAATTTATAATTCTTAATGTCGAGAAGGAGTATCAATGTGTGGCCCAGACTGGTCTTGAACTTTCAATTCACCTGCTGCTCCTCCTCCCCCCTTGCCCAAAAGCGCTATGGTCTCAGGCCTGTGTCAACACACCCAACTAATACTATTAAATCAACAGATAAGTGAGTGATGCAAGGAAATCACTTTACCATCAAGCCTCCCAAAACCCTTTTCGTAGTTTTCCCACGTTTGGTTGAAGTTTTGAGAGCCATCTTTCCGGTGTTGAATTAATGTCCGTGGAGTGCCTGATTGGGTGTCACAGTAGACATTAAACACTTGAGAGCTGCTTGGTCTAATAGTATACACGCCACTTGTATGTTCACCTCTGTTATAAATGGCAGAGCAGTCAGCAGGCAGATCATCTTGTTCTATATTTTTTGCTTCCTTCAGATGAAGAGGGGGAGTAGTTCTTGGTGCTCTTGGTTTAGAATAAAGAGAATTTTCAGTGGGTTCTTGAATGCCAGTCTTTCTGAGCTGATTTTCTATTTCTTTTATCTGAATGTGCTGTTGACTTAGTTGTTTATATTGTTCTTCCACACTCTGGAGGAGTTCTCTTATGCTGTTATCTTGCTGTTCTACAAAACTTTTAAGTGACGTTACCTCTGGGTGCTCCCGAGCCCCAGGCGGGTTCTGAACCAAGCTGGTCAGCTGTTCCTCCAAAGCCCTGACTCTGTGTTGGAGCGCCATCTTCTCCTCCAGTAGACTTTCAAGCTTTGAGTTCAGTTCAAGTGACATATTCTTCACCTCTTCGTTTTTAACTTGTAGTTTAGATGTGGTTCTTCTTAGCTCCTTTTCCTCTTCTTTGATTTCATTGGTTTGAAGTGATAGGTCATAAAAACACTGATCAAATATGTTGAGCTTCTGAAATATGTCATTAATTTGTCCCTTTGTCTTATGGACAAAATCTTTAAGACCATGACCCAGCTGCAGGAGGCCATTGGCTAAAATTTTGACATCATCCAACATAGCAAATCTTGATTTTGGCTCTGACGGTACAGAATCAAATGGCGAAAGGTCTGGATCAACTCTGGACGAAATTACTAGAGGAACAACAAAAAGGAGCAGCTTAATTGTGTGCATTTTTGTTTCAATTATTCAATTTCAAGCAATTTGGAACGTC Macaca fascicularis angiopoietin-like 3 (Angpt13), mRNASEQ ID NO: 9GGGTAGTATATAGAGTTAAGAAGTCTAGGTCTGCTTCCAGAAGAACACAGTTCCACGCTGCTTGAAATTGAAAATCAGGATAAAAATGTTCACAATTAAGCTCCTTCTTTTTATTGTTCCTCTAGTTATTTCCTCCAGAATTGACCAAGACAATTCATCATTTGATTCTGTATCTCCAGAGCCAAAATCAAGATTTGCTATGTTAGACGATGTAAAAATTTTAGCCAATGGCCTCCTTCAGTTGGGACATGGTCTTAAAGACTTTGTCCATAAGACTAAGGGCCAAATTAATGACATATTTCAAAAACTCAACATATTTGATCAGTCTTTTTATGATCTATCACTGCAAACCAGTGAAATCAAAGAAGAAGAAAAGGAACTGAGAAGAACTACATATAAACTACAAGTCAAAAATGAAGAGGTAAAGAATATGTCACTTGAACTCAACTCAAAACTTGAAAGCCTCCTAGAAGAAAAAATTCTACTTCAACAAAAAGTGAAATATTTAGAAGAGCAACTAACTAACTTAATTCAAAATCAACCTGCAACTCCAGAACATCCAGAAGTAACTTCACTTAAAAGTTTTGTAGAAAAACAAGATAATAGCATCAAAGACCTTCTCCAGACTGTGGAAGAACAATATAAGCAATTAAACCAACAGCATAGTCAAATAAAAGAAATAGAAAATCAGCTCAGAATGACTAATATTCAAGAACCCACAGAAATTTCTCTATCTTCCAAGCCAAGAGCACCAAGAACTACTCCCTTTCTTCAGCTGAATGAAATAAGAAATGTAAAACATGATGGCATTCCTGCTGATTGTACCACCATTTACAATAGAGGTGAACATATAAGTGGCACGTATGCCATCAGACCCAGCAACTCTCAAGTTTTTCATGTCTACTGTGATGTTGTATCAGGTAGTCCATGGACATTAATTCAACATCGAATAGATGGATCACAAAACTTCAATGAAACGTGGGAGAACTACAAATATGGTTTCGGGAGGCTTGATGGAGAATTCTGGTTGGGCCTAGAGAAGATATACTCCATAGTGAAGCAATCTAATTACGTTTTACGAATTGAGTTGGAAGACTGGAAAGACAACAAACATTATATTGAATATTCTTTTTACTTGGGAAATCACGAAACCAACTATACGCTACATGTAGTTAAGATTACTGGCAATGTCCCCAATGCAATCCCGGAAAACAAAGATTTGGTGTTTTCTACTTGGGATCACAAAGCAAAAGGACACTTCAGCTGTCCAGAGAGTTATTCAGGAGGCTGGTGGTGGCATGATGAGTGTGGAGAAAACAACCTAAATGGTAAATATAACAAACCAAGAACAAAATCTAAGCCAGAGCGGAGAAGAGGATTATCCTGGAAGTCTCAAAATGGAAGGTTATACTCTATAAAATCAACCAAAATGTTGATCCATCCAACAGATTCAGAAAGCTTTGAATGAACTGAGGCAAATTTAAAAGGCAATAAATTAAACATTAAACTCATTCCAAGTTAATGTGGTTTAATAATCTGGTATTAAATCCTTAAGAGAAGGCTTGAGAAATAGATTTTTTTATCTTAAAGTCACTGTCAATTTAAGATTAAACATACAATCACATAACCTTAAAGAATACCATTTACATTTCTCAATCAAAATTCTTACAACACTATTTGTTTTATATTTTGTGATGTGGGAATCAATTTTAGATGGTCGCAATCTAAATTATAATCAACAGGTGAACTTACTAAATAACTTTTCTAAATAAAAAACTTAGAGACTTTAATTTTAAAAGTCATCATATGAGCTAATGTCACAATTTTCCCAGTTTAAAAAACTAGTTTTCTTGTTAAAACTCTAAACTTGACTAAATAAAGAGGACTGATAATTATACAGTTCTTAAATTTGTTGTAATATTAATTTCAAAACTAAAAATTGTCAGCACAGAGTATGTGTAAAAATCTGTAATATAAATTTTTAAACTGATGCCTCATTTTGCTACAAAATAATCTGGAGTAAATTTTTGATAGGATTTATTTATGAAACCTAATGAAGCAGGATTAAATACTGTATTAAAATAGGTTCGCTGTCTTTTAAACAAATGGAGATGATGATTACTAAGTCACATTGACTTTAATATGAGGTATCACTATACCTTAACATATTTGTTAAAACGTATACTGTATACATTTTGTGT

We claim:
 1. A double-stranded ribonucleic acid (dsRNA) agent forinhibiting expression of Angiopoietin-like 3 (ANGPTL3), comprising asense strand and an antisense strand forming a duplex region, whereinthe sense strand comprises at least 15 contiguous nucleotides of thenucleotide sequence 5′-GUCUCAAAAUGGAAGGUUAUA-3′ (SEQ ID NO: 1160),wherein the antisense strand comprises at least 15 contiguousnucleotides of the nucleotide sequence 5′-UAUAACCUUCCAUUUUGAGACUU-3′(SEQ ID NO: 1341), wherein all of the nucleotides of the sense strandand all of the nucleotides of the antisense strand are modifiednucleotides, wherein at least one of the modified nucleotides isselected from the group consisting of a 2′-O-methyl modified nucleotide,a 2′-fluoro modified nucleotide, and a nucleotide comprising a5′-phosphorothioate group, and wherein a ligand comprising anN-acetylgalactosamine (GalNAc) is conjugated to at least one strand ofthe dsRNA agent.
 2. A double-stranded ribonucleic acid (dsRNA) agent forinhibiting expression of Angiopoietin-like 3 (ANGPTL3), comprising asense strand and an antisense strand forming a duplex region, whereinthe sense strand comprises at least 16 contiguous nucleotides of thenucleotide sequence 5′-GUCUCAAAAUGGAAGGUUAUA-3′ (SEQ ID NO: 1160),wherein the antisense strand comprises at least 16 contiguousnucleotides of the nucleotide sequence 5′-UAUAACCUUCCAUUUUGAGACUU-3′(SEQ ID NO: 1341), wherein all of the nucleotides of the sense strandand all of the nucleotides of the antisense strand are modifiednucleotides, wherein at least one of the modified nucleotides isselected from the group consisting of a 2′-O-methyl modified nucleotide,a 2′-fluoro modified nucleotide, a nucleotide comprising a5′-phosphorothioate group, and wherein a ligand comprising anN-acetylgalactosamine (GalNAc) is conjugated to at least one strand ofthe dsRNA agent.
 3. A double-stranded ribonucleic acid (dsRNA) agent forinhibiting expression of Angiopoietin-like 3 (ANGPTL3), comprising asense strand and an antisense strand forming a duplex region, whereinthe sense strand comprises at least 17 contiguous nucleotides of thenucleotide sequence 5′-GUCUCAAAAUGGAAGGUUAUA-3′ (SEQ ID NO: 1160),wherein the antisense strand comprises at least 17 contiguousnucleotides of the nucleotide sequence 5′-UAUAACCUUCCAUUUUGAGACUU-3′(SEQ ID NO: 1341), wherein all of the nucleotides of the sense strandand all of the nucleotides of the antisense strand are modifiednucleotides, wherein at least one of the modified nucleotides isselected from the group consisting of a 2′-O-methyl modified nucleotideand a 2′-fluoro modified nucleotide, and wherein a ligand comprising anN-acetylgalactosamine (GalNAc) is conjugated to at least one strand ofthe dsRNA agent.
 4. The dsRNA agent of claim 1, wherein the duplexregion is 15-30 base pairs in length.
 5. The dsRNA agent of claim 1,wherein the duplex region is 18-21 base pairs in length.
 6. The dsRNAagent of claim 1, wherein the duplex region is 20 base pairs in length.7. The dsRNA agent of claim 1, wherein the dsRNA agent comprises atleast one phosphorothioate or methylphosphonate internucleotide linkage.8. The dsRNA of claim 1, wherein the GalNAc (N-acetylgalactosamine) isconjugated to at least one strand of the dsRNA agent through a linker.9. The dsRNA of claim 8, wherein the GalNAc (N-acetylgalactosamine) isconjugated through a bivalent or trivalent branched linker.
 10. ThedsRNA of claim 8, wherein the linker comprises C(O)NH.
 11. The dsRNA ofclaim 10, wherein the linker comprises between 1-24 atoms.
 12. The dsRNAof claim 10, wherein the linker comprises between 6-24 atoms.
 13. ThedsRNA agent of claim 1, wherein the sense strand comprises thenucleotide sequence 5′-GUCUCAAAAUGGAAGGUUAUA-3′ (SEQ ID NO: 1160) andthe antisense strand comprises the nucleotide sequence5′-UAUAACCUUCCAUUUUGAGACUU-3′ (SEQ ID NO: 1341).
 14. A cell containingthe dsRNA agent of claim
 1. 15. A pharmaceutical composition forinhibiting expression of an ANGPTL3 gene, comprising the dsRNA agent ofclaim
 1. 16. The pharmaceutical composition of claim 15, wherein thedsRNA agent is present in a buffered solution.
 17. A method ofinhibiting ANGPTL3 expression in a cell, the method comprising: (a)contacting the cell with the dsRNA agent of claim 1; and (b) maintainingthe cell produced in step (a) for a time sufficient to obtaindegradation of the mRNA transcript of an ANGPTL3 gene, therebyinhibiting expression of the ANGPTL3 gene in the cell.
 18. The method ofclaim 17, wherein the cell is within a subject.
 19. A method ofinhibiting the expression of ANGPTL3 in a subject, the method comprisingadministering to the subject a therapeutically effective amount of thedsRNA agent of claim 1, thereby inhibiting the expression of ANGPTL3 inthe subject.
 20. A method of treating a subject having a disorder thatwould benefit from reduction in ANGPTL3 expression, comprisingadministering to the subject a therapeutically effective amount of thedsRNA agent of claim 1, thereby treating the subject.
 21. The method ofclaim 20, wherein the disorder is a disorder of lipid metabolism. 22.The method of claim 20, wherein the disorder is selected from the groupconsisting of hypertriglyceridemia, obesity, hyperlipidemia,atherosclerosis, diabetes, cardiovascular disease, and coronary arterydisease.
 23. The method of claim 22, wherein the disorder ishyperlipidemia.
 24. The method of claim 20, further comprisingadministering an additional therapeutic to the subject.
 25. The methodof claim 24, wherein the additional therapeutic is a statin.
 26. Themethod of claim 20, wherein the dsRNA agent is administered at a dose ofabout 0.5 mg/kg to about 50 mg/kg.
 27. The method of claim 20, whereinthe administration of the dsRNA agent to the subject causes a decreasein one or more serum lipid.
 28. The method of claim 20, wherein theadministration of the dsRNA agent to the subject causes a decrease inANGPTL3 protein accumulation.